Benzyl compounds which inhibit leukocyte adhesion mediated by VLA-4

ABSTRACT

Disclosed are compounds which bind VLA-4. Certain of these compounds also inhibit leukocyte adhesion and, in particular, leukocyte adhesion mediated by VLA-4. Such compounds are useful in the treatment of inflammatory diseases in a mammalian patient, e.g., human, wherein the disease may be, for example, asthma, Alzheimer&#39;s disease, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, rheumatoid arthritis, tissue transplantation, tumor metastasis and myocardial ischemia. The compounds can also be administered for the treatment of inflammatory brain diseases such as multiple sclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/127,601,filed on Jul. 31, 1998, now U.S. Pat. No. 6,362,341, which, in turn,claims the benefit of U.S. Provisional Patent Application No.60/112,007, filed Jul. 31, 1997 and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compounds which inhibit leukocyte adhesionand, in particular, leukocyte adhesion mediated by VLA-4.

2. References

The following publications, patents and patent applications are cited inthis application as superscript numbers:

¹ Hemler and Takada, European Patent Application Publication No.330,506, published Aug. 30, 1989

² Elices, et al., Cell, 60:577-584 (1990)

³ Springer, Nature, 346:425-434 (1990)

⁴ Osborn, Cell, 62:3-6 (1990)

⁵ Vedder, et al., Surgery, 106:509 (1989)

⁶ Pretolani, et al., J. Exp. Med., 180:795 (1994)

⁷ Abraham, et al., J. Clin. Invest., 93:776 (1994)

⁸ Mulligan, et al., J. Immunology, 150:2407 (1993)

⁹ Cybulsky, et al., Science, 251:788 (1991)

¹⁰ Li, et al., Arterioscler. Thromb., 13:197 (1993)

¹¹ Sasseville, et al., Am. J. Path., 144:27 (1994)

¹² Yang, et al., Proc. Nat. Acad. Science (USA), 90:10494 (1993)

¹³ Burkly, et al., Diabetes, 43:529 (1994)

¹⁴ Baron, et al., J. Clin. Invest., 93:1700 (1994)

¹⁵ Hamann, et al., J. Immunology, 152:3238 (1994)

¹⁶ Yednock, et al., Nature, 356:63 (1992)

¹⁷ Baron, et al., J. Exp. Med., 177:57 (1993)

¹⁸ van Dinther-Janssen, et al., J. Immunology, 147:4207 (1991)

¹⁹ van Dinther-Janssen, et al., Annals. Rheumatic Dis., 52:672 (1993)

²⁰ Elices, et al., J. Clin. Invest., 93:405 (1994)

²¹ Postigo, et al., J. Clin. Invest., 89:1445 (1991)

²² Paul, et al., Transpl. Proceed., 25:813 (1993)

²³ Okarhara, et al., Can. Res., 54:3233 (1994)

²⁴ Paavonen, et al., Int. J. Can., 58:298 (1994)

²⁵ Schadendorf, et al., J. Path., 170:429 (1993)

²⁶ Bao, et al., Diff., 52:239 (1993)

²⁷ Lauri, et al., British J. Cancer, 68:862 (1993)

²⁸ Kawaguchi, et al., Japanese J. Cancer Res., 83: 1304 (1992)

²⁹ Kogan, et al., U.S. Pat. No. 5,510,332, issued Apr. 23, 1996

³⁰ International Patent Appl. Publication No. WO 96/01644

All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

3. State of the Art

VLA-4 (also referred to as α4β1 integrin and CD49d/CD29), firstidentified by Hemler and Takada¹, is a member of the β1 integrin familyof cell surface receptors, each of which comprises two subunits, an αchain and a β chain. VLA-4 contains an α4 chain and a β1 chain. Thereare at least nine β1 integrins, all sharing the same β1 chain and eachhaving a distinct α chain. These nine receptors all bind a differentcomplement of the various cell matrix molecules, such as fibronectin,laminin, and collagen. VLA-4, for example, binds to fibronectin. VLA-4is unique among β₁ integrins in that it also binds non-matrix moleculesthat are expressed by endothelial and other cells. These non-matrixmolecules include VCAM-1, which is expressed on cytokine-activated humanumbilical vein endothelial cells in culture. Distinct epitopes of VLA-4are responsible for the fibronectin and VCAM-1 binding activities, andeach activity has been shown to be inhibited independently.²

Intercellular adhesion mediated by VLA-4 and other cell surfacereceptors is associated with a number of inflammatory responses. At thesite of an injury or other inflammatory stimulus, activated vascularendothelial cells express molecules that are adhesive for leukocytes.The mechanics of leukocyte adhesion to endothelial cells involves, inpart, the recognition and binding of cell surface receptors onleukocytes to the corresponding cell surface molecules on endothelialcells. Once bound, the leukocytes migrate across the blood vessel wallto enter the injured site and release chemical mediators to combatinfection. For reviews of adhesion receptors of the immune system, see,for example, Springer³ and Osborn⁴.

Inflammatory brain disorders, such as experimental autoimmuneencephalomyelitis (EAE), multiple sclerosis (MS) and meningitis, areexamples of central nervous system disorders in which theendothelium/leukocyte adhesion mechanism results in destruction tootherwise healthy brain tissue. Large numbers of leukocytes migrateacross the blood brain barrier (BBB) in subjects with these inflammatorydiseases. The leukocytes release toxic mediators that cause extensivetissue damage resulting in impaired nerve conduction and paralysis.

In other organ systems, tissue damage also occurs via an adhesionmechanism resulting in migration or activation of leukocytes. Forexample, it has been shown that the initial insult following myocardialischemia to heart tissue can be further complicated by leukocyte entryto the injured tissue causing still further insult (Vedder et al.⁵).Other inflammatory conditions mediated by an adhesion mechanism include,by way of example, asthma⁶⁻⁸, Alzheimer's disease, atherosclerosis⁹⁻¹⁰,AIDS dementia¹¹, diabetes¹²⁻¹⁴ (including acute juvenile onsetdiabetes), inflammatory bowel disease¹⁵ (including ulcerative colitisand Crohn's disease), multiple sclerosis¹⁶⁻¹⁷, rheumatoidarthritis¹⁸⁻²¹, tissue transplantation²², tumor metastasis²³⁻²⁸,meningitis, encephalitis, stroke, and other cerebral traumas, nephritis,retinitis, atopic dermatitis, psoriasis, myocardial ischemia and acuteleukocyte-mediated lung injury such as that which occurs in adultrespiratory distress syndrome.

In view of the above, assays for determining the VLA-4 level in abiological sample containing VLA-4 would be useful, for example, todiagnosis VLA-4 mediated conditions. Additionally, despite theseadvances in the understanding of leukocyte adhesion, the art has onlyrecently addressed the use of inhibitors of adhesion in the treatment ofinflammatory brain diseases and other inflammatory conditions^(29,30).The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

This invention provides compounds which bind to VLA-4. Such compoundscan be used, for example, to assay for the presence of VLA-4 in a sampleand, in pharmaceutical compositions, to inhibit cellular adhesionmediated by VLA-4, for example, binding of VCAM-1 to VLA-4. Thecompounds of this invention have a binding affinity to VLA-4 asexpressed by an IC₅₀ of about 15 μM or less (as measured by Example 95below), which compounds are defined by formula I below:

where

R¹ is selected from the group consisting of alkyl, substituted alkyl,aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heterocyclic, substituted heterocylic, heteroaryl and substitutedheteroaryl;

R² and R³ together with the nitrogen atom bound to R² and the carbonatom bound to R³ form a saturated heterocyclic group or a saturatedsubstituted heterocyclic group with the proviso that whenmonosubstituted, the substituent on said saturated heterocyclic group isnot carboxyl;

R⁵ is selected from the group consisting of —(CH₂)_(n)-aryl and—(CH₂)_(n)-heteroaryl, where n is an integer equal to 1 to 4;

Q is —C(X)NR⁷— wherein R⁷ is selected from the group consisting ofhydrogen and alkyl, and X is selected from the group consisting ofoxygen and sulfur;

and pharmaceutically acceptable salts thereof,

with the proviso that when R¹ is 2,4,6-trimethylphenyl, R² and R³together with the pendent nitrogen and carbon atoms form a pyrrolidinylring and Q is —C(O)NH—, then R⁵ is not benzyl; and

with the further proviso that when R¹ is p-methylphenyl, R² and R³together with the pendent nitrogen and carbon atoms form a pyrrolidinylring derived from D-proline and Q is —C(O)NH—, then R⁵ is not benzylderived from D-phenylalanine.

In another embodiment, the compounds of this invention can also beprovided as prodrugs which convert (e.g., hydrolyze, metabolize, etc.)in vivo to a compound of formula I above. In a preferred example of suchan embodiment, the carboxylic acid of the compound of formula I ismodified into a group which, in vivo, will convert to a carboxylic acid(including salts thereof). In a particularly preferred embodiment, suchprodrugs are represented by compounds of formula IA:

R¹ is selected from the group consisting of alkyl, substituted alkyl,aryl, substituted aryl, cycloalkyl, heterocyclic, substitutedheterocylic, heteroaryl and substituted heteroaryl;

R² and R³ together with the nitrogen atom bound to R² and the carbonatom bound to R³ form a saturated heterocyclic group or a saturatedsubstituted heterocyclic group with the proviso that whenmonosubstituted, the substituent on said saturated heterocyclic group isnot carboxyl;

R⁵ is selected from the group consisting of —(CH₂)_(n)-aryl and—(CH₂)_(n)-heteroaryl, where n is an integer equal to 1 to 4;

R⁶ is selected from the group consisting of amino, alkoxy, substitutedalkoxy, cycloalkoxy, substituted cycloalkoxy, —O-(N-succinimidyl),—NH-adamantyl, —O-cholest-5-en-3-β-yl, —NHOY where Y is hydrogen, alkyl,substituted alkyl, aryl, and substituted aryl, —NH(CH₂)_(p)COOY where pis an integer of from 1 to 8 and Y is as defined above, —OCH₂NR⁹R¹⁰where R⁹ is selected from the group consisting of —C(O)-aryl and—C(O)-substituted aryl and R¹⁰ is selected from the group consisting ofhydrogen and —CH₂COOR¹¹ where R¹¹ is alkyl, and —NHSO₂Z where Z isalkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, asubstituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic;

Q is —C(X)NR⁷— wherein R⁷ is selected from the group consisting ofhydrogen and alkyl, and X is selected from the group consisting ofoxygen and sulfur;

and pharmaceutically acceptable salts thereof,

with the following provisos:

A. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a pyrrolidin-2-yl ring, R⁵ is benzyl andQ is —C(O)NH—, then R⁶ is not —NH(CH₂)₂CO₂Et or -(1R,2S,5R)-(−)-menthylester;

B. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a 3-β-phenyl-ring derived from D-proline,R⁵ is benzyl and Q is —C(O)NH—, then R⁶ is not —OCH₂CH₃;

C. when R¹ is 1-N-methyl-3-methyl-5-chloropyrazol-4-yl, R² and R³together with the pendent nitrogen and carbon atoms form apyrrolidin-2-yl ring, R⁵ is benzyl and Q is —C(O)NH—, then R⁶ is not—OCH₃;

D. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a pyrrolidin-2-yl ring, R⁵ is D-benzyland Q is —C(O)NH—, then R⁶ is not —OCH₂CH₃;

E. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a 5,5-dimethyl-1,1-dioxo-thiaprolyl ring,R⁵ is benzyl and Q is —C(O)NH—, then R⁶ is not —OC(CH₃)₃; and

F. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a pyrrolidin-2-yl ring derived fromD-proline, R⁵ is benzyl derived from D-phenylalanine and Q is —C(O)NH—,then R⁶ is not —OCH₃;

G. when R¹ is n-butyl, R² and R³ together with the pendent nitrogen andcarbon atoms form a pyrrolidin-2-yl ring, R⁵ is benzyl and Q is—C(O)NH—, then R⁶ is not —O-benzyl.

Preferably, in the compounds of formula I and IA above, R¹ is selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, heterocyclic, substituted heterocylic, heteroaryl and substitutedheteroaryl. Even more preferably R¹ is selected from the groupconsisting of 4-methylphenyl, methyl, benzyl, n-butyl, 4-chlorophenyl,1-naphthyl, 2-naphthyl, 4-methoxyphenyl, phenyl, 2,4,6-trimethylphenyl,2-(methoxycarbonyl)phenyl, 2-carboxyphenyl, 3,5-dichlorophenyl,4-trifluoromethylphenyl, 3,4-dirhlorophenyl, 3,4-dimethoxyphenyl,4-(CH₃C(O)NH-)phenyl, 4-trifluoromethoxyphenyl, 4-cyanophenyl,isopropyl, 3,5-di-(trifluoromethyl)phenyl, 4-t-butylphenyl,4-t-butoxyphenyl, 4-nitrophenyl, 2-thienyl,1-N-methyl-3-methyl-5-chloropyrazol-4-yl, phenethyl,1-N-methylimidazol-4-yl, 4-bromophenyl, 4-amidinophenyl,4-methylamidinophenyl, 4-[CH₃SC(═NH)]phenyl, 5-chloro-2-thienyl,2,5-dichloro-4-thienyl, 1-N-methyl-4-pyrazolyl, 2-thiazolyl,5-methyl-1,3,4-thiadiazol-2-yl, 4-[H₂NC(S)]phenyl, 4-aminophenyl,4-fluorophenyl, 2-fluorophenyl, 3-fluorophenyl, 3,5-difluorophenyl,pyridin-3-yl, pyrimidin-2-yl, and 4-(3′-dimethylamino-n-propoxy)-phenyl.

In one preferred embodiment, R² and R³ together with the nitrogen atombound to the R² substituent and the carbon bound to the R³ substituentform a heterocyclic group or substituted heterocyclic group of 4 to 6ring atoms having 1 to 2 heteroatoms in the ring selected from nitrogen,oxygen and sulfur, which ring is optionally substituted with 1 to 2substituents selected from fluoro, methyl, hydroxy, amino, phenyl,thiophenyl and thiobenzyl, or can be fused to another saturatedheterocyclic or cycloalkyl ring such as a cyclohexyl ring to provide fora fused ring heterocycle of from 10 to 14 ring atoms having 1 to 2heteroatoms in the ring selected from nitrogen, oxygen and sulfur. Suchheterocyclic rings include thiazolidinyl (e.g., L-thiazolidin-4-yl),piperidinyl (e.g., L-piperidin-2-yl), piperizinyl (e.g.,L-piperizin-2-yl), thiomorpholinyl (e.g., L-thiomorpholin-3-yl),pyrrolidinyl (e.g., L-pyrrolidin-2-yl), substituted pyrrolidinyl such as4-hydroxypyrrolidinyl (e.g., 4-α-(or β-)hydroxy-L-pyrrolidin-2-yl),4-fluoropyrrolidinyl (e.g., 4-α-(or β-)fluoro-L-pyrrolidin-2-yl),3-phenylpyrrolidinyl (e.g., 3-α-(or β-)phenyl-L-pyrrolidin-2-yl),3-thiophenylpyrrolidinyl (e.g., 3-α-(orβ-)thiophenyl-L-pyrrolidin-2-yl), 4-aminopyrrolidinyl (e.g., 4-α-(orβ-)amino-L-pyrrolidin-2-yl), 3-methoxypyrrolidinyl (e.g., 3-α-(orβ-)methoxy-L-pyrrolidin-2-yl), 4,4-dimethylpyrrolidin-2-yl, substitutedpiperizinyl such as 4-N-Cbz-piperizin-2-yl, substituted thiazolidinylsuch as 5,5-dimethylthiazolindin-4-yl, 1,1-dioxothiazolidinyl (e.g.,L-1,1-dioxo-thiazolidin-4-yl), substituted 1,1-dioxothiazolidinyl suchas L-1,1-dioxo-5,5-dimethylthiazolidin-4-yl, 1,1-dioxothiomorpholinyl(e.g., L-1,1-dioxo-thiomorpholin-3-yl) and the like. Preferably, suchrings do not include those where R² and R³ together with the nitrogenatom bound to R² and the carbon atom bound to R³ form a azetidine ring.

Q is preferably —C(O)NH— or —C(S)NH—.

R⁵ is preferably selected from all possible isomers arising bysubstitution of the following groups: benzyl, phenethyl,—CH₂-(3-indolyl), —CH₂-(1-naphthyl), —CH₂-(2-naphthyl),—CH₂-(2-thienyl), —CH₂-(3-pyridyl), —CH₂-(5-imidazolyl),—CH₂-3-(1,2,4-triazolyl), —CH₂-(2-thiazolyl) and the like.

In the compounds of formula IA, R⁶ is preferably2,4-dioxo-tetrahydrofuran-3-yl (3,4-enol), methoxy, ethoxy, iso-propoxy,n-butoxy, t-butoxy, cyclopentoxy, neo-pentoxy,2-α-iso-propyl-4-β-methylcyclohexoxy,2-β-isopropyl-4-β-methylcyclohexoxy, —NH₂, benzyloxy, —NHCH₂COOH,—NHCH₂CH₂COOH, —NH-adamantyl, —NHCH₂CH₂COOCH₂CH₃, —NHSO₂-p-CH₃-φ, —NHOR⁸where R⁸ is hydrogen, methyl, iso-propyl or benzyl, O-(N-succinimidyl),—O-cholest-5-en-3-β-yl, —OCH₂—OC(O)C(CH₃)₃, —O(CH₂)_(z)NHC(O)W where zis 1 or 2 and W is selected from the group consisting of pyrid-3-yl,N-methylpyridyl, and N-methyl-1,4-dihydro-pyrid-3-yl, and —NR″C(O)—R′where R′ is aryl, heteroaryl or heterocyclic and R″ is hydrogen or—CH₂C(O)OCH₂CH₃.

Preferred compounds within the scope of formula I and IA above includeby way of example:

N-(methanesulfonyl)-L-prolyl-L-phenylalanine

N-(a-toluenesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-(N-methyl)phenylalanine

N-(toluene-4-sulfonyl)-L-pipecolinyl-L-phenylalanine

N-(toluene-4-sulfonyl)-D-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-(4-hydroxy)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-D,L-homophenylalanine

N-(4-chlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(1-naphthalenesulfonyl)-L-prolyl-L-phenylalanine

N-(2-naphthalenelsulfonyl)-L-prolyl-L-phenylalanine

N-(4-methoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-tert-butylbenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-(4-fluoro)prolyl-L-phenylalanine

N-(n-butanesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanine

N-(2-methoxycarbonylbenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(2-carboxybenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-thiaprolyl-L-phenylalanine

N-(3,5-dichlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-trifluoromethoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(3,4-dichlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-D,L-(3-phenyl)prolyl-L-phenylalanine

N-(3,4-dimethoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-nitrobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-acetamidobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-cyanobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-tryptophan

N-(toluene-4-sulfonyl)-L-prolyl-β-(1-naphthyl)-L-alanine

N-(toluene-4-sulfonyl)-L-prolyl-β-(2-naphthyl)-L-alanine

N-(toluene-4-sulfonyl)-L-prolyl-β-(2-thienyl)-L-alanine

N-(isopropanesulfonyl)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-β-(3-pyridyl)-L-alanine

N-(toluene-4-sulfonyl)-L-(4-phenylthio)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-(4-benzylthio)-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-histidine

N-(toluene-4-sulfonyl)-L-(4-amino)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalaninamide

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine benzyl ester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine ethyl ester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine N-methoxyamide

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine N-benzyloxyamide

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-(toluene-4-sulfonyl)amide

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalaninyl-β-alanine

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine N-hydroxyamide

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine isopropyl ester

N-(toluene-4-sulfonyl)-L-(4-hydroxy)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalaninyl-(N-benzoyl)glycineethyl ester

N-(toluene-4-sulfonyl)-L-(4-fluoro)prolyl-L-phenylalanine benzyl ester

N-(toluene-4-sulfonyl)-L-thiaprolyl-L-phenylalanine benzyl ester

N-(toluene-4-sulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanine benzylester

N-(toluene-4-sulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanine ethylester

N-(2-methoxycarbonylbenzenesulfonyl)-L-prolyl-L-phenylalanine benzylester

N-(toluene-4-sulfonyl)-L-(3-phenyl)prolyl-L-phenylalanine ethyl ester

N-(toluene-4-sulfonyl)-L-(4-methoxy)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine (1S,2R,5S)-(+)-menthylester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine N-hydroxysuccinimideester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine 2-(nicotinamido)ethylester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine2-(1-methylpyridinium-3-amido)ethyl ester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine cholesteryl ester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine2-(1-methyl-1,4-dihydropyridinyl-3-amido)ethyl ester

N-(thiophene-2-sulfonyl)-L-prolyl-L-phenylalanine methyl ester

N-(thiophene-2-sulfonyl)-L-prolyl-L-phenylalanine

N-(5-chloro-1,3-dimethylpyrazole-4-sulfonyl)-L-prolyl-L-phenylalanine

N-(2-phenylethanesulfonyl)-L-prolyl-L-phenylalanine

N-(1-methylimidazole-4-sulfonyl)-L-prolyl-L-phenylalanine methyl ester

N-(1-methylimidazole-4-sulfonyl)-L-prolyl-L-phenylalanine

N-(4-amidinobenzenesulfonyl)-L-prolyl-L-phenylalanine methyl ester

N-(4-amidinobenzenesulfonyl)-L-prolyl-L-phenylalanine

N-(4-thiomethoxyimidatylbenzene-4-sulfonyl)-L-prolyl-L-phenylalaninemethyl ester

N-[4-(N-methylthioamido)benzenesulfonyl]-L-prolyl-L-phenylalanine methylester

N-(toluene-4-sulfonyl)-L-prolyl-D,L-β-(1,2,4-triazol-3-yl)alanine

N-(toluene-4-sulfonyl)-L-prolyl-D,L-β-(thiazol-2-yl)alanine

N-[4-(3-dimethylaminopropyloxy)benzenesulfonyl]-L-prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-pyrrolidin-2-yl-thiocarbonyl-L-phenylalanine

N-(4-thiocarbamoylbenzenesulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalaninebenzyl ester

N-(4-cyanobenzenesulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalaninebenzyl ester

N-(toluene-4-sulfonyl)-L-prolyl-D-phenylalanine

N-(toluene-4-sulfonyl)-L-(thiamorpholin-3-carbonyl)-L-phenylalanine

N-(toluene-4-sulfonyl)-[(1,1-dioxo)thiamorpholin-3-carbonyl]-L-phenylalanine

N-(toluene-4-sulfonyl)-L-(3,3-dimethyl)prolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-L-(5,5-dimethyl-1,1-dioxo)thiaprolyl-L-phenylalanine

N-(toluene-4-sulfonyl)-[(1,1-dioxo)thiamorpholin-3-carbonyl]-L-phenylalanineethyl ester

N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine tert-butyl ester

N-(toluene-4-sulfonyl)-L-pyrrolidin-2-yl-thiocarbonyl-L-phenylalaninemethyl ester

N-(benzenesulfonyl)-L-prolyl-L-phenylalanine methyl ester

N-(toluene-4-sulfonyl)-L-(5-oxo)prolyl-L-phenylalanine ethyl ester

N-(toluene-4-sulfonyl)-L-(5-oxo)prolyl-L-phenylalanine

and pharmaceutically acceptable salts thereof, as well as any of theester compounds recited above wherein one ester is replaced with anotherester selected from the group consisting of methyl ester, ethyl ester,n-propyl ester, isopropyl ester, n-butyl ester, isobutyl ester,sec-butyl ester and tert-butyl ester.

This invention also provides methods for binding VLA-4 in a biologicalsample which method comprises contacting the biological sample with acompound of formula I or IA above under conditions wherein said compoundbinds to VLA-4.

Certain of the compounds of formula I and IA above are also useful inreducing VLA-4 mediated inflammation in vivo.

This invention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of one or more of the compounds of formula I or IA above, withthe exception that R³ and R⁵ are derived from L-amino acids or othersimilarly configured starting materials. Alternatively, racemic mixturescan be used.

The pharmaceutical compositions may be used to treat VLA-4 mediateddisease conditions. Such disease conditions include, by way of example,asthma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes(including acute juvenile onset diabetes), inflammatory bowel disease(including ulcerative colitis and Crohn's disease), multiple sclerosis,rheumatoid arthritis, tissue transplantation, tumor metastasis,meningitis, encephalitis, stroke, and other cerebral traumas, nephritis,retinitis, atopic dermatitis, psoriasis, myocardial ischemia and acuteleukocyte-mediated lung injury such as that which occurs in adultrespiratory distress syndrome.

Accordingly, this invention also provides methods for the treatment ofan inflammatory disease in a patient mediated by VLA-4 which methodscomprise administering to the patient the pharmaceutical compositionsdescribed above.

Preferred compounds of formula I and IA above include those set forth inTable I below:

TABLE I I

Q = —C(O)NR⁷— R¹ R² R³ R⁵ R^(6′) R⁷ H₃C— R²/R³ = cyclic —CH₂-φ —OH H 3carbon atoms (L-pyrrolidinyl) φ-CH₂— R²/R³ = cyclic —CH₂-φ —OH H 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH —CH₃ 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H 4carbon atoms (L-piperidin-2-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H 3carbon atoms (D-pyrrolidinyl) p-CH₃-φ- R²/R³ = —CH₂-φ —OH H—CH₂CH(OH)CH₂— (L-4-β-hydroxy- pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H 2 carbon aloms (L-isomer) p-CH₃-φ- R²/R³ = cyclic —CH₂CH₂-φ—OH H 3 carbon atoms (L-pyrrolidinyl) p-Cl-φ- R²/R³ = cyclic —CH₂-φ —OHH 3 carbon atoms (L-pyrrolidinyl) 1-naphthyl- R²/R³ = cyclic —CH₂-φ —OHH 3 carbon atoms (L-pyrrolidinyl) 2-naphthyl- R²/R³ = cyclic —CH₂-φ —OHH 3 carbon atoms (L-pyrrolidinyl) p-CH₃O-φ- R²/R³ = cyclic —CH₂-φ —OH H3 carbon atoms (L-pyrrolidinyl) p-(t-butyl)-φ- R²/R³ = cyclic —CH₂-φ —OHH 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H—CH₂CH(F)CH₂— (4-β-fluoro-L- pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H —CH₂CH(F)CH₂— (4-α-fluoro-L-pyrrolidinyl) n-butyl R²/R³ =cyclic —CH₂-φ —OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic —CH₂-φ —OH H —CH₂S—C(CH₃)₂— (L-5,5-dimethylthiazolidin-4-yl)o-methoxy- R²/R³ = cyclic —CH₂-φ —OH H carbonyl-φ- 3 carbon atoms(L-pyrrolidinyl) o-carboxyl-φ- R²/R³ = cyclic —CH₂-φ —OH H 3 carbonatoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H —CH₂SCH₂—(L-thiazolidin-4-yl) 3,5-dichloro- R²/R³ = cyclic —CH₂-φ —OH H φ- 3carbon atoms (L-pyrrolidinyl) p-trifluoro- R²/R³ = cyclic —CH₂-φ —OH Hmethoxy-φ- 3 carbon atoms (L-pyrrolidinyl) 3,4-dichloro- R²/R³ = cyclic—CH₂-φ —OH H φ- 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H —CH₂CH₂CH(φ)— (3-β-phenyl-L-pyrrolidin-2-yl) 3,4- R²/R³ =cyclic —CH₂-φ —OH H dimethoxy-φ- 3 carbon atoms (L-pyrrolidinyl) φ-R²/R³ = cyclic —CH₂-φ —OH H —CH₂CH₂CH(φ)— (3-β-phenyl-L-pyrrolidinyl)p-nitro-φ- R²/R³ = cyclic —CH₂-φ —OH H 3 carbon atoms (L-pyrrolidinyl)p- R²/R³ = cyclic —CH₂-φ —OH H CH₃C(O)NH- 3 carbon atoms φ-(L-pyrrolidinyl) p-cyano-φ- R²/R³ = cyclic —CH₂-φ —OH H 3 carbon atoms(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic (3-indolyl)-CH₂— —OH H 3 carbonatoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-(1-naphthyl) —OH H 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-(2-naphthyl)—OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-(2-thienyl) —OH H 3 carbon atoms (L-pyrrolidinyl) isopropyl R²/R³ =cyclic —CH₂φ —OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic 3-pyridyl-CH₂— —OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ-R²/R³ = cyclic —CH₂-φ —OH H —CH₂CH(Sφ)CH₂—(3-α-thienyl-L-pyrrolidinyl-2-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H—CH₂CH(SCH₂φ)CH₂— 3-(α-thienyl-D-pyrrolidinyl-2-yl) p-CH₃-φ- R²/R³ =cyclic imidazol-5-yl-CH₂— —OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ-R²/R³ = cyclic —CH₂-φ —OH H —CH₂CH(NH₂)CH₂—(4-α-amino-L-pyrrolidin-2-yl) p-CH₃-φ- R²/R³ = 4-β-amino-D- —CH₂-φ —OH Hpyrrolidin-2-yl p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —NH₂ H 3 carbon atoms(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O—CH₂-φ H 3 carbonatoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O-ethyl H 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —NHOCH₃ H 3carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —NHO—CH₂-φH 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ—NHSO₂-(p- H 3 carbon atoms methylphenyl) (L-pyrrolidinyl) p-CH₃-φ-R²/R³ = cyclic —CH₂-φ —NH(CH₂)₂—CO₂H H 3 carbon atoms (L-pyrrolidinyl)p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —NHOH H 3 carbon atoms (L-pyrrolidinyl)p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O-isopropyl H 3 carbon atoms(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H —CH₂CH(OH)CH₂—(4-α-hydroxy-D-pyrrolidin-2-yl) p-CH₃-φ- R²/R³ = cyclic 3 carbon atoms(L-pyrrolidinyl) —CH₂-φ

H p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O—CH₂-φ H —CH₂CH(F)CH₂—(4-β-fluoro-L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O—CH₂-φ H—CH₂CH(F)CH₂— (4-α-fluoro-L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ—O—CH₂-φ H —CH₂S—CH₂— (L-thiazolidin-4-yl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —O—CH₂-φ H —CH₂S—C(CH₃)₂— (L-5,5-dimethylthiazolidin-4-yl)p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₃ H —CH₂S—C(CH₃)₂—(L-5,5-dimethylthiazolidin-4-yl) methoxycar- R²/R³ = cyclic —CH₂-φ—O—CH₂-φ H bonyl-φ 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic —CH₂-φ —OCH₂CH₃ H —CH₂CH₂CH(φ)— (3-β-phenyl-L-pyrrolidin-2-yl)p-CH₃-φ- R²/R³ = cyclic —CH₂-φ OH H —CH₂CH(OCH₃)CH₂—(3-methoxy-L-pyrrolidin-2-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ2-β-isopropyl-4-α- H 3 carbon atoms methyl-cyclohex-1- (L-pyrrolidinyl)oxy p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O—(N- H 3 carbon atoms succinimidyl)(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₂— H 3 carbonatoms NHC(O)- (L-pyrrolidinyl) pyrid-3-yl p-CH₃-φ- R²/R³ = cyclic —CH₂-φ—OCH₂CH₂— H 3 carbon atoms NHC(O)—N- (L-pyrrolidinyl) methylpyrid-3-ylp-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O-cholest-5-en-3- H 3 carbon atoms β-yl(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₂— H 3 carbonatoms NHC(O)— (L-pyrrolidinyl) N-methyl-1,4- dihydro-pyrid-3-yl2-thienyl R²/R³ = cyclic —CH₂-φ —OCH₃ H 3 carbon atoms (L-pyrrolidinyl)2-thienyl R²/R³ = cyclic —CH₂-φ —OH H 3 carbon atoms (L-pyrrolidinyl)1-N-methyl-3- R²/R³ = cyclic —CH₂-φ —OH H methyl-5- 3 carbon atomschloropyrazol- (L-pyrrolidinyl) 4-yl φ-CH₂CH₂— R²/R³ = cyclic —CH₂-φ —OHH 3 carbon atoms (L-pyrrolidinyl) 1-N-methyl- R²/R³ = cyclic —CH₂-φ—OCH₃ H imidazol-4-yl 3 carbon atoms (L-pyrrolidinyl) 1-N-methyl- R²/R³= cyclic —CH₂-φ —OH H imidazol-4-yl 3 carbon atoms (L-pyrrolidinyl)

R²/R³ = cyclic 3 carbon atoms (L-pyrrolidinyl) —CH₂-φ —OCH₃ H

R²/R³ = cyclic 3 carbon atoms (L-pyrrolidinyl) —CH₂-φ —OH H

R²/R³ = cyclic 3 carbon atoms (L-pyrrolidinyl) —CH₂-φ —OCH₃ H

R²/R³ = cyclic 3 carbon atoms (L-pyrrolidinyl) —CH₂-φ —OCH₃ H p-CH₃-φ-R²/R³ = cyclic —CH₂-3-(1,2,4-triazolyl) —OH H 3 carbon atoms(L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-2-thiazolyl —OH H 3 carbonatoms (L-pyrrolidinyl) p-[-O(CH₂)₃— R²/R³ = cyclic —CH₂-φ —OH HN(CH₃)₂]-φ- 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH Q = —C(S)NH— 3 carbon atoms (L-pyrrolidinyl) p-NH₂(S)C-φ-R²/R³ = cyclic —CH₂-φ —OCH₂-φ H —CH₂—S—C(CH₃)₂—(L-5,5-dimethylthiazolidin-4-yl) p-NC-φ- R²/R³ = cyclic —CH₂-φ —OCH₂-φ H—CH₂—S—C(CH₃)₂— (L-5,5-dimethylthiazolidin-4-yl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H —CH₂CH₂—S—CH₂CH₂— (L-5,5-dimethylthiazolidin-4-yl) p-CH₃-φ-R²/R³ = cyclic —CH₂-φ —OH H —CH₂CH₂—SO₂—CH₂— (L-1,1-dioxothiomorpholin-3-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H—CH₂—CH₂—C(CH₃)₂— (L-4,4-dimethylpyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OH H —CH₂—SO₂—C(CH₃)₂— (L-1,1-dioxo-5,5-dimethylthiazolidin-4-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₃ H—CH₂CH₂—SO₂—CH₂— (L-1,1-dioxothiomorpholin-3-yl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OC(CH₃)₃ H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic —CH₂-φ —OCH₃ Q = —C(S)NH— 3 carbon atoms (L-pyrrolidinyl) φ-R²/R³ = cyclic —CH₂-φ —OCH₃ H 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ-R²/R³ = cyclic —CH₂-φ —OCH₂CH₃ H —C(O)—CH₂—CH₂— (L-5-oxopyrrolidinyl)p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H —C(O)—CH₂—CH₂—(L-5-oxopyrrolidinyl)

DETAILED DESCRIPTION OF THE INVENTION

As above, this invention relates to compounds which inhibit leukocyteadhesion and, in particular, leukocyte adhesion mediated by VLA-4.However, prior to describing this invention in further detail, thefollowing terms will first be defined.

Definitions

As used herein, “alkyl” refers to alkyl groups preferably having from 1to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term isexemplified by groups such as methyl, t-butyl, n-heptyl, octyl and thelike.

“Substituted alkyl” refers to an alkyl group, preferably of from 1 to 10carbon atoms, having from 1 to 5 substituents selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino,thiocarbonylamino, acyloxy, amino, amidino, alkyl amidino, thioamidino,aminoacyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aryl, substituted aryl, aryloxy, substituted aryloxy, aryloxylaryl,substituted aryloxyaryl, cyano, halogen, hydroxyl, nitro, carboxyl,carboxylalkyl, carboxyl-substituted alkyl, carboxyl-cycloalkyl,carboxyl-substituted cycloalkyl, carboxylaryl, carboxyl-substitutedaryl, carboxylheteroaryl, carboxyl-substituted heteroaryl,carboxylheterocyclic, carboxyl-substituted heterocyclic, cycloalkyl,substituted cycloalkyl, guanidino, guanidinosulfone, thiol, thioalkyl,substituted thioalkyl, thioaryl, substituted thioaryl, thiocycloalkyl,substituted thiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic,cycloalkoxy, substituted cycloalkoxy, heteroaryloxy, substitutedheteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR whereR is hydrogen or alkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl,—NRS(O)₂-aryl, —NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl,—NRS(O)₂-substituted heteroaryl, —NRS(O)₂-heterocyclic,—NRS(O)₂-substituted heterocyclic, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl, —NRS(O)₂—NR-substitutedaryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR-substituted heteroaryl,—NRS(O)₂—NR-heterocyclic, —NRS(O)₂—NR-substituted heterocyclic where Ris hydrogen or alkyl, mono- and di-alkylamino, mono- and di-(substitutedalkyl)amino, mono- and di-arylamino, mono- and di-substituted arylamino,mono- and di-heteroarylamino, mono- and di-substituted heteroarylamino,mono- and di-heterocyclic amino, mono- and di-substituted heterocyclicamino, unsymmetric di-substituted amines having different substituentsselected from alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic, and substituted alkyl groups having amino groups blockedby conventional blocking groups such as Boc, Cbz, formyl, and the like,and alkyl/substituted alkyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

“Alkoxy” refers to the group “alkyl-O—” which includes, by way ofexample, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

“Substituted alkoxy” refers to the group “substituted alkyl-O—”.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)-cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O), heterocyclic-C(O)—, and substituted heterocyclic-C(O)—,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Acylamino” refers to the group —C(O)NRR where each R is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic, andwhere each R is joined to form, together with the nitrogen atom, aheterocyclic or substituted heterocyclic ring, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Thiocarbonylamino” refers to the group —C(S)NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, and where each R is joined to form, together with thenitrogen atom, a heterocyclic or substituted heterocyclic ring, whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substitutedheteroaryl-C(O)O—, heterocyclic-C(O)O—, and substitutedheterocyclic-C(O)O—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Alkenyl” refers to an alkenyl group preferably having from 2 to 10carbon atoms, and more preferably 2 to 6 carbon atoms, and having atleast 1, and preferably from 1-2, sites of alkenyl unsaturation.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy, substituted aryloxy, aryloxyaryl, substituted aryloxyaryl,halogen, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxylcycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic and—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituents selectedfrom alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andsubstituted alkenyl groups having amino groups blocked by conventionalblocking groups such as Boc, Cbz, formyl, and the like, andalkenyl/substituted alkenyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

“Alkynyl” refers to an alkynyl group preferably having from 2 to 10carbon atoms, and more preferably 3 to 6 carbon atoms, and having atleast 1, and preferably from 1-2, sites of alkynyl unsaturation.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, acyl, acylamino, thiocarbonylamino, acyloxy, amino, amidino,alkylamidino, thioamidino, aminoacyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aryl, substituted aryl,aryloxy. substituted aryloxy, aryloxyaryl, substituted aryloxyaryl,halogen, hydroxyl, cyano, nitro, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxylcycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, thiol, thioalkyl, substituted thioalkyl,thioaryl, substituted thioaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheteroaryl, substituted thioheteroaryl,thioheterocyclic, substituted thioheterocyclic, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, oxycarbonylamino,oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-substituted alkyl,—OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic and—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituents selectedfrom alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andsubstituted alkynyl groups having amino groups blocked by conventionalblocking groups such as Boc, Cbz, formyl, and the like, andalkynyl/substituted alkynyl groups substituted with —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

“Amidino” refers to the group

and the term “alkylamidino” refers to compounds having 1 to 3 alkylgroups

“Thioamidino” refers to the group

where R is hydrogen or alkyl.

“Aminoacyl” refers to the groups —NRC(O)alkyl, —NRC(O)substituted alkyl,—NRC(O)cycloalkyl, —NRC(O)substituted cycloalkyl, —NRC(O)alkenyl,—NRC(O)substituted alkenyl, —NRC(O)alkynyl, —NRC(O)substituted alkynyl,—NRC(O)aryl, —NRC(O)substituted aryl, —NRC(O)heteroaryl,—NRC(O)substituted heteroaryl, —NRC(O)heterocyclic, and—NRC(O)substituted heterocyclic where R is hydrogen or alkyl, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the groups —NRC(O)O-alkyl,—NRC(O)O-substituted alkyl, —NRC(O)O-alkenyl, —NRC(O)O-substitutedalkenyl, —NRC(O)O-alkynyl, —NRC(O)O-substituted alkynyl,—NRC(O)O-cycloalkyl, —NRC(O)O-substituted cycloalkyl, —NRC(O)O-aryl,—NRC(O)O-substituted aryl, —NRC(O)O-heteroaryl, —NRC(O)O-substitutedheteroaryl, —NRC(O)O-heterocyclic, and —NRC(O)O-substituted-heterocyclicwhere R is hydrogen or alkyl, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Oxycarbonylamino” refers to the groups —OC(O)NH₂, —OC(O)NRR,—OC(O)NR-alkyl, —OC(O)NR-substituted alkyl, —OC(O)NR-alkenyl,—OC(O)NR-substituted alkenyl, —OC(O)NR-alkynyl, —OC(O)NR-substitutedalkynyl, —OC(O)NR-cycloalkyl, —OC(O)NR-substituted cycloalkyl,—OC(O)NR-aryl, —OC(O)NR-substituted aryl, —OC(O)NR-heteroaryl,—OC(O)NR-substituted heteroaryl , —OC(O)NR-heterocyclic, and—OC(O)NR-substituted heterocyclic where R is hydrogen, alkyl or whereeach R is joined to form, together with the nitrogen atom, aheterocyclic or substituted heterocyclic ring, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Oxythiocarbonylamino” refers to the groups —OC(S)NH₂, —OC(S)NRR,—OC(S)NR-alkyl, —OC(S)NR-substituted alkyl, —OC(S)NR-alkenyl,—OC(S)NR-substituted alkenyl, —OC(S)NR-alkynyl, —OC(S)NR-substitutedalkynyl, —OC(S)NR-cycloalkyl, —OC(S)NR-substituted cycloalkyl,—OC(S)NR-aryl, —OC(S)NR-substituted aryl, —OC(S)NR-heteroaryl,—OC(S)NR-substituted heteroaryl, —OC(S)NR-heterocyclic, and—OC(S)NR-substituted heterocyclic where R is hydrogen or alkyl, or whereeach R is joined to form, together with the nitrogen atom, aheterocyclic or substituted heterocyclic ring, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Aminocarbonylamino” refers to the groups —NRC(O)NRR, —NRC(O)NR-alkyl,—NRC(O)NR-substituted alkyl, —NRC(O)NR-alkenyl, —NRC(O)NR-substitutedalkenyl, —NRC(O)NR-alkynyl, —NRC(O)NR-substituted alkynyl,—NRC(O)NR-aryl, —NRC(O)NR-substituted aryl, —NRC(O)NR-cycloalkyl,—NRC(O)NR-substituted cycloalkyl, —NRC(O)NR-heteroaryl,—NRC(O)NR-substituted heteroaryl, —NRC(O)NR-heterocyclic, and—NRC(O)NR-substituted heterocyclic where each R is independentlyhydrogen or alkyl, or where each R is joined to form, together with thenitrogen atom, a heterocyclic or substituted heterocyclic ring, as wellas where one of the amino groups is blocked by conventional blockinggroups such as Boc, Cbz, formyl, and the like, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic and substitutedheterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the groups —NRC(S)NRR,—NRC(S)NR-alkyl, —NRC(S)NR-substituted alkyl, —NRC(S)NR-alkenyl,—NRC(S)NR-substituted alkenyl, —NRC(S)NR-alkynyl, —NRC(S)NR-substitutedalkynyl, —NRC(S)NR-aryl, —NRC(S)NR-substituted aryl,—NRC(S)NR-cycloalkyl, —NRC(S)NR-substituted cycloalkyl,—NRC(S)NR-heteroaryl, —NRC(S)NR-substituted heteroaryl,—NRC(S)NR-heterocyclic, and —NRC(S)NR-substituted heterocyclic whereeach R is independently hydrogen or alkyl, or where each R is joined toform, together with the nitrogen atom, a heterocyclic or substitutedheterocyclic ring, as well as where one of the amino groups is blockedby conventional blocking groups such as Boc, Cbz, formyl, and the like,and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Aryl” or “Ar” refers to an unsaturated aromatic carbocyclic group offrom 6 to 14 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl), which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7yl, and the like). Preferred aryls includephenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with from1 to 3 substituents selected from the group consisting of hydroxy, acyl,acylamino, thiocarbonylamino, acyloxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, amidino, alkylamidino, thioamidino, amino, aminoacyl,aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino, aryl,substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy, substitutedcycloalkoxy, heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy,substituted heterocyclyloxy, carboxyl, carboxylalkyl,carboxyl-substituted alkyl, carboxyl-cycloalkyl, carboxyl-substitutedcycloalkyl, carboxylaryl, carboxyl-substituted aryl, carboxylheteroaryl,carboxyl-substituted heteroaryl, carboxylheterocyclic,carboxyl-substituted heterocyclic, carboxylamido, cyano, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substitutedthiocycloalkyl, thioheterocyclic, substituted thioheterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, halo,nitro, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, oxycarbonylamino, oxythiocarbonylamino, —S(O)₂-alkyl,—S(O)₂-substituted alkyl, —S(O)₂-cycloalkyl, —S(O)₂-substitutedcycloalkyl, —S(O)₂-alkenyl, —S(O)₂-substituted alkenyl, —S(O)₂-aryl,—S(O),-substituted aryl, —S(O)₂-heteroaryl, —S(O)₂-substitutedheteroaryl, —S(O)₂-heterocyclic, —S(O)₂-substituted heterocyclic,—OS(O)₂-alkyl, —OS(O)₂-substituted alkyl, —OS(O)₂-aryl,—OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl, —OS(O)₂-substitutedheteroaryl, —OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic,—OSO₂—NRR where R is hydrogen or alkyl, —NRS(O)-alkyl,—NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl, —NRS(O)₂-substituted aryl,—NRS(O)₂-heteroaryl, —NRS(O)₂-substituted heteroaryl,—NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic and—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituents selectedfrom alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andamino groups on the substituted aryl blocked by conventional blockinggroups such as Boc, Cbz, formyl, and the like, or substituted with—SO₂NRR where R is hydrogen or alkyl.

“Aryloxy” refers to the group aryl-O— which includes, by way of example,phenoxy, naphthoxy, and the like.

“Substituted aryloxy” refers to substituted aryl-O— groups.

“Aryloxyaryl” refers to the group -aryl-O-aryl.

“Substituted aryloxyaryl” refers to aryloxyaryl groups substituted withfrom 1 to 3 substituents on either or both aryl rings selected from thegroup consisting of hydroxy, acyl, acylamino, thiocarbonylamino,acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic and—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituents selectedfrom alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andamino groups on the substituted aryl blocked by conventional blockinggroups such as Boc, Cbz, formyl, and the like, or substituted with—SO₂NRR where R is hydrogen or alkyl.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 8 carbon atomshaving a single cyclic ring including, by way of example, cyclopropyl,cyclobutyl, cyclopentyl, cycloactyl and the like. Excluded from thisdefinition are multi-ring alkyl groups such as adamantanyl, etc.

“Cycloalkenyl” refers to cyclic alkenyl groups of from 3 to 8 carbonatoms having single or multiple unsaturation but which are not aromatic.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refer tocycloalkyl and cycloalkenyl groups, preferably of from 3 to 8 carbonatoms, having from 1 to 5 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy, acyl,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, substitutedthioheteroaryl, thioheterocyclic, substituted thioheterocyclic,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR whereR is hydrogen or alkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl,—NRS(O)₂-aryl, —NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl,—NRS(O)₂-substituted heteroaryl, —NRS(O)₂-heterocyclic,—NRS(O)₂-substituted heterocyclic, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted alkyl, —NkS(O)₂—NR-aryl, —NRS(O)₂—NR-substitutedaryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR-substituted heteroaryl,—NRS(O)₂—NR-heterocyclic and —NRS(O)₂—NR-substituted heterocyclic whereR is hydrogen or alkyl, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-substituted arylamino, mono- and di-heteroarylamino, mono- anddi-substituted heteroarylamino, mono- and di-heterocyclic amino, mono-and di-substituted heterocyclic amino, unsymmetric di-substituted amineshaving different substituents selected from alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclicand substituted heterocyclic, and substituted alkynyl groups havingamino groups blocked by conventional blocking groups such as Boc, Cbz,formyl, and the like, and alkynyl/substituted alkynyl groups substitutedwith —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substitutedalkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

“Cycloalkoxy” refers to —O-cycloalkyl groups.

“Substituted cycloalkoxy” refers to —O-substituted cycloalkyl groups.

“Guanidino” refers to the groups —NRC(═NR)NRR, —NRC(═NR)NR-alkyl,—NRC(═NR)NR-substituted alkyl, —NRC(═NR)NR-alkenyl,—NRC(═NR)NR-substituted alkenyl, —NRC(═NR)NR-alkynyl,—NRC(═NR)NR-substituted alkynyl, —NRC(═NR)NR-aryl,—NRC(═NR)NR-substituted aryl, —NRC(═NR)NR-cycloalkyl,—NRC(═NR)NR-substituted cycloalkyl, —NRC(═NR)NR-heteroaryl,—NRC(═NR)NR-substituted heteroaryl, —NRC(═NR)NR-heterocyclic, and—NRC(═NR)NR-substituted heterocyclic where each R is independentlyhydrogen and alkyl, as well as where one of the amino groups is blockedby conventional blocking groups such as Boc, Cbz, formyl, and the like,and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic andsubstituted heterocyclic are as defined herein.

“Guanidinosulfone” refers to the groups —NRC(═NR)NRSO₂-alkyl,—NRC(═NR)NRSO₂-substituted alkyl, —NRC(═NR)NRSO₂-alkenyl,—NRC(═NR)NRSO₂-substituted alkenyl, —NRC(═NR)NRSO₂-alkynyl,—NRC(═NR)NRSO₂-substituted alkynyl, —NRC(═NR)NRSO₂-aryl,—NRC(═NR)NRSO₂-substituted aryl, —NRC(═NR)NRSO₂-cycloalkyl,—NRC(═NR)NRSO₂-substituted cycloalkyl, —NRC(═NR)NRSO₂-heteroaryl,—NRC(═NR)NRSO₂-substituted heteroaryl, —NRC(═NR)NRSO₂-heterocyclic, and—NRC(═NR)NRSO₂-substituted heterocyclic where each R is independentlyhydrogen and alkyl, and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is either chloro or bromo.

“Heteroaryl” refers to an aromatic carbocyclic group of from 2 to 10carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen andsulfur within the ring. Such heteroaryl groups can have a single ring(e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinylor benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl,indolyl and furyl.

“Substituted heteroaryl” refers to heteroaryl groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of hydroxy, acyl, acylamino, thiocarbonylamino, acyloxy,alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, amidino,alkylamidino, thioamidino, amino, aminoacyl, aminocarbonyloxy,aminocarbonylamino, aminothiocarbonylamino, aryl, substituted aryl,aryloxy, substituted aryloxy, cycloalkoxy, substituted cycloalkoxy,heteroaryloxy, substituted heteroaryloxy, heterocyclyloxy, substitutedheterocyclyloxy, carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,carboxylamido, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl,substituted thioaryl, thioheteroaryl, substituted thioheteroaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic,substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl,guanidino, guanidinosulfone, halo, nitro, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, oxycarbonylamino,oxythiocarbonylamino, —S(O)₂-alkyl, —S(O)₂-substituted alkyl,—S(O)₂-cycloalkyl, —S(O)₂-substituted cycloalkyl, —S(O)₂-alkenyl,—S(O)₂-substituted alkenyl, —S(O)₂-aryl, —S(O)₂-substituted aryl,—S(O)₂-heteroaryl, —S(O)₂-substituted heteroaryl, —S(O)₂-heterocyclic,—S(O)₂-substituted heterocyclic, —OS(O)₂-alkyl, —OS(O)₂-substitutedalkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl, —OS(O)₂-heteroaryl,—OS(O)₂-substituted heteroaryl, —OS(O)₂-heterocyclic,—OS(O)₂-substituted heterocyclic, —OSO₂—NRR where R is hydrogen oralkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl, —NRS(O)₂-aryl,—NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl, —NRS(O)₂-substitutedheteroaryl, —NRS(O)₂-heterocyclic, —NRS(O)₂-substituted heterocyclic,—NRS(O)₂—NR-alkyl, —NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl,—NRS(O)₂—NR-substituted aryl, —NRS(O)₂—NR-heteroaryl,—NRS(O)₂—NR-substituted heteroaryl, —NRS(O)₂—NR-heterocyclic and—NRS(O)₂—NR-substituted heterocyclic where R is hydrogen or alkyl, mono-and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-substituted arylamino, mono- anddi-heteroarylamino, mono- and di-substituted heteroarylamino, mono- anddi-heterocyclic amino, mono- and di-substituted heterocyclic amino,unsymmetric di-substituted amines having different substituents selectedfrom alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic and substituted heterocyclic, andamino groups on the substituted aryl blocked by conventional blockinggroups such as Boc, Cbz, formyl, and the like, or substituted with—SO₂NRR where R is hydrogen or alkyl.

“Heteroaryloxy” refers to the group —O-heteroaryl and “substitutedheteroaryloxy” refers to the group —O-substituted heteroaryl.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings having from 1 to10 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen,sulfur or oxygen within the ring wherein, in fused ring systems, one ormore the rings can be aryl or heteroaryl.

“Saturated heterocyclic” refers to heterocycles of single or multiplecondensed rings lacking unsaturation in any ring (e.g., carbon to carbonunsaturation, carbon to nitrogen unsaturation, nitrogen to nitrogenunsaturation, and the like).

“Unsaturated heterocyclic” refers to non-aromatic heterocycles of singleor multiple condensed rings having unsaturation in any ring (e.g.,carbon to carbon unsaturation, carbon to nitrogen unsaturation, nitrogento nitrogen unsaturation, and the like).

“Substituted heterocyclic” refers to heterocycle groups which aresubstituted with from 1 to 3 substituents selected from the groupconsisting of oxo (═O), thioxo (═S), alkoxy, substituted alkoxy, acyl,acylamino, thiocarbonylamino, acyloxy, amino, amidino, alkylamidino,thioamidino, aminoacyl, aminocarbonylamino, aminothiocarbonylamino,aminocarbonyloxy, aryl, substituted aryl, aryloxy, substituted aryloxy,aryloxyaryl, substituted aryloxyaryl, halogen, hydroxyl, cyano, nitro,carboxyl, carboxylalkyl, carboxyl-substituted alkyl,carboxyl-cycloalkyl, carboxyl-substituted cycloalkyl, carboxylaryl,carboxyl-substituted aryl, carboxylheteroaryl, carboxyl-substitutedheteroaryl, carboxylheterocyclic, carboxyl-substituted heterocyclic,cycloalkyl, substituted cycloalkyl, guanidino, guanidinosulfone, thiol,thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl,thiocycloalkyl, substituted thiocycloalkyl, thioheteroaryl, substitutedthioheteroaryl, thioheterocyclic, substituted thioheterocyclic,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, —OSO₂—NRR whereR is hydrogen or alkyl, —NRS(O)₂-alkyl, —NRS(O)₂-substituted alkyl,—NRS(O)₂-aryl, —NRS(O)₂-substituted aryl, —NRS(O)₂-heteroaryl,—NRS(O)₂-substituted heteroaryl, —NRS(O)₂-heterocyclic,—NRS(O)₂-substituted heterocyclic, —NRS(O)₂—NR-alkyl,—NRS(O)₂—NR-substituted alkyl, —NRS(O)₂—NR-aryl, —NRS(O)₂—NR-substitutedaryl, —NRS(O)₂—NR-heteroaryl, —NRS(O)₂—NR-substituted heteroaryl,—NRS(O)₂—NR-heterocyclic and —NRS(O)₂—NR-substituted heterocyclic whereR is hydrogen or alkyl, mono- and di-alkylamino, mono- anddi-(substituted alkyl)amino, mono- and di-arylamino, mono- anddi-substituted arylamino, mono- and di-heteroarylamino, mono- anddi-substituted heteroarylamino, mono- and di-heterocyclic amino, mono-and di-substituted heterocyclic amino, unsymmetric di-substituted amineshaving different substituents selected from alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclicand substituted heterocyclic, and substituted alkynyl groups havingamino groups blocked by conventional blocking groups such as Boc, Cbz,formyl, and the like, and alkynyl/substituted alkynyl groups substitutedwith —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substitutedalkenyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic and —SO₂NRR where R ishydrogen or alkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholino, thiomorpholino, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

“Saturated substituted heterocyclic” refers to substituted heterocyclesof single or multiple condensed rings lacking unsaturation in any ring(e.g., carbon to carbon unsaturation, carbon to nitrogen unsaturation,nitrogen to nitrogen unsaturation, and the like).

“Unsaturated substituted heterocyclic” refers to non-aromaticsubstituted heterocycles of single or multiple condensed rings havingunsaturation in any ring (e.g., carbon to-carbon unsaturation, carbon tonitrogen unsaturation, nitrogen to nitrogen unsaturation, and the like).

“Heterocyclyloxy” refers to the group —O-heterocyclic and “substitutedheterocyclyloxy” refers to the group —O-substituted heterocyclic.

“Thiol” refers to the group —SH.

“Thioalkyl” refers to the groups —S-alkyl

“Substituted thioalkyl” refers to the group —S-substituted alkyl.

“Thiocycloalkyl” refers to the groups —S-cycloalkyl.

“Substituted thiocycloalkyl” refers to the group —S-substitutedcycloalkyl.

“Thioaryl” refers to the group —S-aryl and “substituted thioaryl” refersto the group —S-substituted aryl.

“Thioheteroaryl” refers to the group —S-heteroaryl and “substitutedthioheteroaryl” refers to the group —S-substituted heteroaryl.

“Thioheterocyclic” refers to the group —S-heterocyclic and “substitutedthioheterocyclic” refers to the group —S-substituted heterocyclic.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound of formula I or IA, which salts are derived from avariety of organic and inorganic counter ions well known in the art andinclude, by way of example only, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium, and the like; and when the compound offormula I or IA contains a basic functionality, salts of organic orinorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like.

Compound Preparation

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

Furthermore, the compounds of this invention will typically contain oneor more chiral centers. Accordingly, if desired, such compounds can beprepared or isolated as pure stereoisomers i.e., as individualenantiomers or diastereomers, or as stereoisomer-enriched mixtures. Allsuch stereoisomers (and enriched mixtures) are included within the scopeof this invention, unless otherwise indicated. Pure stereoisomers (orenriched mixtures) may be prepared using, for example, optically activestarting materials or stereoselective reagents well-known in the art.Alternatively, racemic mixtures of such compounds can be separatedusing, for example, chiral column chromatography, chiral resolvingagents and the like.

In a preferred method of synthesis, the compounds of formulas I and IA,wherein Q is —C(O)NR⁷—, are prepared by first coupling an amino acid offormula II:

wherein R² and R³ are as defined in formulas I and IA, and R⁴ ishydrogen, with a sulfonyl chloride of formula III:

wherein R¹ is as defined in formulas I and IA, to provide an N-sulfonylamino acid of formula IV:

wherein R¹-R⁴ are as defined above.

This reaction is typically conducted by reacting the amino acid offormula II with at least one equivalent, preferably about 1.1 to about 2equivalents, of sulfonyl chloride III in an inert diluent such asdichloromethane and the like. Generally, the reaction is conducted at atemperature ranging from about −70° C. to about 40° C. for about 1 toabout 24 hours. Preferably, this reaction is conducted in the presenceof a suitable base to scavenge the acid generated during the reaction.Suitable bases include, by way of example, tertiary amines, such astriethylamine, diisopropylethylamine, N-methylmorpholine and the like.Alternatively, the reaction can be conducted under Schotten-Baumann-typeconditions using aqueous alkali, such as sodium hydroxide and the like,as the base. Upon completion of the reaction, the resuling N-sulfonylamino acid IV is recovered by conventional methods includingneutralization, extraction, precipitation, chromatography, filtrationand the like.

The amino acids of formula II employed in the above reaction are eitherknown compounds or compounds that can be prepared from known compoundsby conventional synthetic procedures. Examples of suitable amino acidsfor use in this reaction include, but are not limited to, L-proline,trans-4-hydroxyl-L-proline, cis-4-hydroxyl-L-proline,trans-3-phenyl-L-proline, cis-3-phenyl-L-proline, L-(2-methyl)proline,L-pipecolinic acid, L-indoline-2-carboxylic acid,L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,L-thiazolidine-4-carboxylic acid,L-(5,5-dimethyl)thiazolidine-4-carboxylic acid,L-thiamorpholine-3-carboxylic acid, glycine, 2-tert-butylglycine,D,L-phenylglycine, L-alanine, α-methylalanine, N-methyl-L-phenylalanine,L-diphenylalanine, sarcosine, D,L-phenylsarcosine, L-aspartic acidβ-tert-butyl ester, L-glutamic acid γ-tert-butyl ester,L-(O-benzyl)serine, 1-aminocyclopropanecarboxylic acid,1-aminocyclobutanecarboxylic acid, 1-aminocyclopentanecarboxylic acid(cycloleucine) 1-aminocyclohexanecarboxylic acid, L-serine and the like.If desired, the corresponding carboxylic acid esters of the amino acidsof formula II, such as the methyl esters, ethyl esters and the like, canbe employed in the above reaction with the sulfonyl chloride III.Subsequent hydrolysis of the ester group to the carboxylic acid usingconventional reagents and conditions, i.e., treatment with an alkalimetal hydroxide in an inert diluent such as methanol/water, thenprovides the N-sulfonyl amino acid IV.

Similarly, the sulfonyl chlorides of formula III employed in the abovereaction are either known compounds or compounds that can be preparedfrom known compounds by conventional synthetic procedures. Suchcompounds are typically prepared from the corresponding sulfonic acid,i.e., from compounds of the formula R¹—SO₃H where R¹ is as definedabove, using phosphorous trichloride and phosphorous pentachloride. Thisreaction is generally conducted by contacting the sulfonic acid withabout 2 to 5 molar equivalents of phosphorous trichloride andphosphorous pentachloride, either neat or in an inert solvent, such asdichloromethane, at a temperature in the range of about 0° C. to about80° C. for about 1 to about 48 hours to afford the sulfonyl chloride.Alternatively, the sulfonyl chlorides of formula III can be preparedfrom the corresponding thiol compound, i.e., from compounds of theformula R¹—SH where R¹ is as defined above, by treating the thiol withchlorine (Cl₂) and water under conventional reaction conditions.

Examples of sulfonyl chlorides suitable for use in this inventioninclude, but are not limited to, methanesulfonyl chloride,2-propanesulfonyl chloride, 1-butanesulfonyl chloride, benzenesulfonylchloride, 1-naphthalenesulfonyl chloride, 2-naphthalenesulfonylchloride, p-toluenesulfonyl chloride, α-toluenesulfonyl chloride,4-acetamidobenzenesulfonyl chloride, 4-amidinobenzenesulfonyl chloride,4-tert-butylbenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride,2-carboxybenzenesulfonyl chloride, 4-cyanobenzenesulfonyl chloride,3,4-dichlorobenzenesulfonyl chloride, 3,5-dichlorobenzenesulfonylchloride, 3,4-dimethoxybenzenesulfonyl chloride,3,5-ditrifluoromethylbenzenesulfonyl chloride, 4-fluorobenzenesulfonylchloride, 4-methoxybenzenesulfonyl chloride,2-methoxycarbonylbenzenesulfonyl chloride, 4-methylamidobenzenesulfonylchloride, 4-nitrobenzenesulfonyl chloride, 4-thioamidobenzenesulfonylchloride, 4-trifluoromethylbenzenesulfonyl chloride,4-trifluoromethoxybenzenesulfonyl chloride,2,4,6-trimethylbenzenesulfonyl chloride, 2-phenylethanesulfonylchloride, 2-thiophenesulfonyl chloride, 5-chloro-2-thiophenesulfonylchloride, 2,5-dichloro-4-thiophenesulfonyl chloride, 2-thiazolesulfonylchloride, 2-methyl4-thiazolesulfonyl chloride,1-methyl-4-imidazolesulfonyl chloride, 1-methyl4-pyrazolesulfonylchloride, 5-chloro-1,3-dimethyl-4-pyrazolesulfonyl chloride,3-pyridinesulfonyl chloride, 2-pyrimidinesulfonyl chloride and the like.If desired, a sulfonyl fluoride, sulfonyl bromide or sulfonic acidanhydride may be used in place of the sulfonyl chloride in the abovereaction to form the N-sulfonyl amino acids of formula IV.

The compounds of formula I are then prepared by coupling theintermediate N-sulfonyl amino acid of formula IV with an amino acidderivative of formula VI:

wherein R⁵-R⁷ are as in formulas I and IA. This coupling reaction istypically conducted using well-known coupling reagents such ascarbodiimides, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphonate) and the like. Suitable carbodiimides include, byway of example, dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and the like. Ifdesired, polymer supported forms of carbodiimide coupling reagents mayalso be used including, for example, those described in TetrahedronLetters, 34(48), 7685 (1993). Additionally, well-known couplingpromoters, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, may be used to facilitate the coupling reaction.

This coupling reaction is typically conducted by contacting theN-sulfonylamino acid IV with about 1 to about 2 equivalents of thecoupling reagent and at least one equivalent, preferably about 1 toabout 1.2 equivalents, of amino acid derivative VI in an inert diluent,such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like. Generally, this reaction isconducted at a temperature ranging from about 0° C. to about 37° C. forabout 12 to about 24 hours. Upon completion of the reaction, thecompound of formula I is recovered by conventional methods includingneutralization, extraction, precipitation, chromatography, filtration,and the like.

Alternatively, the N-sulfonyl amino acid IV can be converted into anacid halide and the acid halide coupled with amino acid derivative VI toprovide compounds of formula I. The acid halide of VI can be prepared bycontacting VI with an inorganic acid halide, such as thionyl chloride,phosphorous trichloride, phosphorous tribromide or phosphorouspentachloride, or, preferably, with oxalyl chloride under conventionalconditions. Generally, this reaction is conducted using about 1 to 5molar equivalents of the inorganic acid halide or oxalyl chloride,either neat or in an inert solvent, such as dichloromethane or carbontetrachloride, at temperature in the range of about 0° C. to about 80°C. for about 1 to about 48 hours. A catalyst, such asN,N-dimethylformamide, may also be used in this reaction.

The acid halide of N-sulfonyl amino acid IV is then contacted with atleast one equivalent, preferably about 1.1 to about 1.5 equivalents, ofamino acid derivative VI in an inert diluent, such as dichloromethane,at a temperature ranging from about −70° C. to about 40° C. for about 1to about 24 hours. Preferably, this reaction is conducted in thepresence of a suitable base to scavenge the acid generated during thereaction. Suitable bases include, by way of example, tertiary amines,such as triethylamine, diisopropylethylamine, N-methylmorpholine and thelike. Alternatively, the reaction can be conducted underSchotten-Baumann-type conditions using aqueous alkali, such as sodiumhydroxide and the like. Upon completion of the reaction, the compound offormula I is recovered by conventional methods including neutralization,extraction, precipitation, chromatography, filtration, and the like.

Alternatively, the compounds of formula I can be prepared by firstforming a diamino acid derivative of formula VII:

wherein R²-R⁷ are as defined above. The diamino acid derivatives offormula VII can be readily prepared by coupling an amino acid of formulaII with an amino acid derivative of formula VI using conventional aminoacid coupling techniques and reagents, such as carbodiimides, BOPreagent and the like, as described above. Diamino acid VII can then besulfonated using a sulfonyl chloride of formula III and using thesynthetic procedures described above to provide a compound of formula I.

The amino acid derivatives of formula VI employed in the above reactionsare either known compounds or compounds that can be prepared from knowncompounds by conventional synthetic procedures. For example, amino acidderivatives of formula VI can De prepared by C-alkylating commerciallyavailable diethyl 2-acetamidomalonate (Aldrich, Milwaukee, Wis., USA)with an alkyl or substituted alkyl halide. This reaction is typicallyconducted by treating the diethyl 2-acetamidomalonate with at least oneequivalent of sodium ethoxide and at least one equivalent of an alkyl orsubstituted alkyl halide in refluxing ethanol for about 6 to about 12hours. The resulting C-alkylated malonate is then deacetylated,hydrolyzed and decarboxylated by heating in aqueous hydrochloric acid atreflux for about 6 to about 12 hours to provide the amino acid,typically as the hydrochloride salt.

Examples of amino acid derivatives of formula VI suitable for use in theabove reactions include, but are not limited to, L-tryptophan methylester, L-phenylalanine methyl ester, L-phenylalanine isopropyl ester,L-phenylalanine benzyl ester, L-phenylalaninamide,N-methyl-L-phenylalanine benzyl ester, D,L-homophenylalanine methylester, β-(1-naphthyl)-L-alanine methyl ester, β-(2-naphthyl)-L-alaninemethyl ester, β-(2-thienyl)-L-alanine methyl ester,β-(2-pyridyl)-L-alanine methyl ester, β-(3-pyridyl)-L-alanine methylester, β-(4-pyridyl)-L-alanine methyl ester, β-(2-thiazolyl)-D,L-alaninemethyl ester, β-(1,2,4-triazol-3-yl)-D,L-alanine methyl ester, and thelike. If desired, of course, other esters or amides of theabove-described compounds may also be employed.

For ease of synthesis, the compounds of formula I are typically preparedas an ester, i.e., where R⁶ is an alkoxy or substituted alkoxy group andthe like. If desired, the ester group can be hydrolysed usingconventional conditions and reagents to provide the correspondingcarboxylic acid. Typically, this reaction is conducted by treating theester with at least one equivalent of an alkali metal hydroxide, such aslithium, sodium or potassium hydroxide, in an inert diluent, such asmethanol or mixtures of methanol and water, at a temperature rangingfrom about 0° C. to about 24° C. for about 1 to about 12 hours.Alternatively, benzyl esters may be removed by hydrogenolysis using apalladium catalyst, such as palladium on carbon. The resultingcarboxylic acids may be coupled, if desired, to amines such as β-alanineethyl ester, hydroxyamines such as hydroxylamine andN-hydroxysuccinimide, alkoxyamines and substituted alkoxyamines such asO-methylhydroxylamine and O-benzylhydroxylamine, and the like, usingconventional coupling reagents and conditions as described above.

As will be apparent to those skilled in the art, other functional groupspresent on any of the substituents of the compounds of formula I can bereadily modified or derivatized either before or after theabove-described coupling reactions using well-known syntheticprocedures. For example, a nitro group present on a substituent of acompound of formula I or an intermediate thereof may be readily reducedby hydrogenation in the presence of a palladium catalyst, such aspalladium on carbon, to provide the corresponding amino group. Thisreaction is typically conducted at a temperature of from about 20° C. toabout 50° C. for about 6 to about 24 hours in an inert diluent, such asmethanol. Compounds having a nitro group on the R⁵ substituent can beprepared, for example, by using a 4-nitrophenylalanine derivative andthe like in the above-described coupling reactions.

Similarly, a pyridyl group can be hydrogenated in the presence of aplatinum catalyst, such as platinum oxide, in an acidic diluent toprovide the corresponding piperidinyl analogue. Generally, this reactionis conducted by treating the pyridine compound with hydrogen at apressure ranging from about 20 psi to about 60 psi, preferably about 40psi, in the presence of the catalyst at a temperature of about 20° C. toabout 50° C. for about 2 to about 24 hours in an acidic diluent, such asa mixture of methanol and aqueous hydrochloric acid. Compounds having apyridyl group can be readily prepared by using, for example,β-(2-pyridyl)-, β-(3-pyridyl)- or β-(4-pyridyl)-L-alanine derivatives inthe above-described coupling reactions.

By way of illustration, a compound of formula I, or an intermediatethereof, having a substituent containing a primary or secondary aminogroup, such as where R¹ is a 4-aminophenyl group, can be readilyN-acylated using conventional acylating reagents and conditions toprovide the corresponding amide. This acylation reaction is typicallyconducted by treating the amino compound with at least one equivalent,preferably about 1.1 to about 1.2 equivalents, of a carboxylic acid inthe presence of a coupling reagent such as a carbodiimide, BOP reagent(benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphonate) and the like, in an inert diluent, such asdichloromethane, chloroform, acetonitrile, tetrahydrofuran,N,N-dimethylformamide and the like, at a temperature ranging from about0° C. to about 37° C. for about 4 to about 24 hours. Preferably, apromoter, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and thelike, is used to facilitate the acylation reaction. Examples ofcarboxylic acids suitable for use in this reaction include, but are notlimited to, N-tert-butyloxycarbonylglycine,N-tert-butyloxycarbonyl-L-phenylalanine,N-tert-butyloxycarbonyl-L-aspartic acid benzyl ester, benzoic acid,N-tert-butyloxycarbonylisonipecotic acid, N-methylisonipecotic acid,N-tert-butyloxycarbonylnipecotic acid,N-tert-butyloxycarbonyl-L-tetrahydroisoquinoline-3-carboxylic acid,N-(toluene-4-sulfonyl)-L-proline and the like.

Alternatively, a compound of formula I or an intermediate thereofcontaining a primary or secondary amino group can be N-acylated using anacyl halide or a carboxylic acid anhydride to form the correspondingamide. This reaction is typically conducted by contacting the aminocompound with at least one equivalent, preferably about 1.1 to about 1.2equivalents, of the acyl halide or carboxylic acid anhydride in an inertdiluent, such as dichloromethane, at a temperature ranging from about−70° C. to about 40° C. for about 1 to about 24 hours. If desired, anacylation catalyst such as 4-(N,N-dimethylamino)pyridine may be used topromote the acylation reaction. The acylation reaction is preferablyconducted in the presence of a suitable base to scavenge the acidgenerated during the reaction. Suitable bases include, by way ofexample, tertiary amines, such as triethylamine, diisopropylethylamine,N-methylmorpholine and the like. Alternatively, the reaction can beconducted under Schotten-Baumann-type conditions using aqueous alkali,such as sodium hydroxide and the like.

Examples of acyl halides and carboxylic acid anhydrides suitable for usein this reaction include, but are not limited to, 2-methylpropionylchloride, trimethylacetyl chloride, phenylacetyl chloride, benzoylchloride, 2-bromobenzoyl chloride, 2-methylbenzoyl chloride,2-trifluoromethylbenzoyl chloride, isonicotinoyl chloride, nicotinoylchloride, picolinoyl chloride, acetic anhydride, succinic anhydride andthe like. Carbamyl chlorides, such as N,N-dimethylcarbamyl chloride,N,N-diethylcarbamyl chloride and the like, can also be used in thisreaction to provide ureas. Similarly, dicarbonates, such asdi-tert-butyl dicarbonate, may be employed to provide carbamates.

In a similar manner, a compound of formula I or an intermediate thereofcontaining a primary or secondary amino group may be N-sulfonated toform a sulfonamide using a sulfonyl halide or a sulfonic acid anhydride.Sulfonyl halides and sulfonic acid anhydrides suitable for use in thisreaction include, but are not limited to, methanesulfonyl chloride,chloromethanesulfonyl chloride, p-toluenesulfonyl chloride,trifluoromethanesulfonic anhydride and the like. Similarly, sulfamoylchlorides, such as dimethylsulfamoyl chloride, can be used to providesulfamides (e.g., >N—SO₂—N<).

Additionally, a primary and secondary amino group present on asubstituent of a compound of formula I, or an intermediate thereof, canbe reacted with an isocyanate or a thioisocyanate to give a urea orthiourea, respectively. This reaction is typically conducted bycontacting the amino compound with at least one equivalent, preferablyabout 1.1 to about 1.2 equivalents, of the isocyanate or thioisocyanatein an inert diluent, such as toluene and the like, at a temperatureranging from about 24° C. to about 37° C. for about 12 to about 24hours. The isocyanates and thioisocyanates used in this reaction arecommercially available or can be prepared from commercially availablecompounds using well-known synthetic procedures. For example,isocyanates and thioisocyanates are readily prepared by reacting theappropriate amine with phosgene or thiophosgene. Examples of isocyanatesand thioisocyanates suitable for use in this reaction include, but arenot limited to, ethyl isocyanate, n-propyl isocyanate, 4-cyanophenylisocyanate, 3-methoxyphenyl isocyanate, 2-phenylethyl isocyanate, methylthioisocyanate, ethyl thioisocyanate, 2-phenylethyl thioisocyanate,3-phenylpropyl thioisocyanate, 3-(N,N-diethylamino)propylthioisocyanate, phenyl thioisocyanate, benzyl thioisocyanate, 3-pyridylthioisocyanate, fluorescein isothiocyanate (isomer I), and the like.

Furthermore, when a compound of formula I or an intermediate thereofcontains a primary or secondary amino group, the amino group can bereductively alkylated using aldehydes or ketones to form a secondary ortertiary amino group. This reaction is typically conducted by contactingthe amino compound with at least one equivalent, preferably about 1.1 toabout 1.5 equivalents, of an aldehyde or ketone and at least oneequivalent based on the amino compound of a metal hydride reducingagent, such as sodium cyanoborohydride, in an inert diluent, such asmethanol, tetrahydrofuran, mixtures thereof and the like, at atemperature ranging from about 0° C. to about 50° C. for about 1 toabout 72 hours. Aldehydes and ketones suitable for use in this reactioninclude, by way of example, benzaldehyde, 4-chlorobenzaldehyde,valeraldehyde and the like.

In a similar manner, when a compound of formula I, or an intermediatethereof, has a substituent containing a hydroxyl group, the hydroxylgroup can be further modified or derivatized either before or after theabove coupling reactions to provide, by way of example, ethers,carbamates and the like.

By way of example, a compound of formula I or an intermediate thereofhaving a substituent containing a hydroxyl group, such as where R¹ is a4-hydroxyphenyl group, can be readily O-alkylated to form ethers. ThisO-alkylation reaction is typically conducted by contacting the hydroxycompound with a suitable alkali or alkaline earth metal base, such aspotassium carbonate, in an inert diluent, such as acetone, 2-butanoneand the like, to form the alkali or alkaline earth metal salt of thehydroxyl group. This salt is generally not isolated, but is reacted insitu with at least one equivalent of an alkyl or substituted alkylhalide or sulfonate, such as an alkyl chloride, bromide, iodide,mesylate or tosylate, to afford the ether. Generally, this reaction isconducted at a temperature ranging from about 60° C. to about 150° C.for about 24 to about 72 hours. Preferably, a catalytic amount of sodiumor potassium iodide is added to the reaction mixture when an alkylchloride or bromide is employed in the reaction.

Examples of alkyl or substituted alkyl halides and sulfonates suitablefor use in this reaction include, but are not limited to, tert-butylbromoacetate, N-tert-butyl chloroacetamide, 1-bromoethylbenzene, ethylα-bromophenylacetate, 2-(N-ethyl-N-phenylamino)ethyl chloride,2-(N,N-ethylamino)ethyl chloride, 2-(N,N-diisopropylamino)ethylchloride, 2-(N,N-dibenzylamino)ethyl chloride, 3-(N,N-ethylamino)propylchloride, 3-(N-benzyl-N-methylamino)propyl chloride,N-(2-chloroethyl)morpholine, 2-(hexamethyleneimino)ethyl chloride,3-(N-methylpiperazine)propyl chloride,1-(3-chlorophenyl)4-(3-chloropropyl)piperazine,2-(4-hydroxy-4-phenylpiperidine)ethyl chloride,N-tert-butyloxycarbonyl-3-piperidinemethyl tosylate and the like.

Alternatively, a hydroxyl group present on a substituent of a compoundof formula I, or an intermediate thereof, can be O-alkylated using theMitsunobu reaction. In this reaction, an alcohol, such as3-(N,N-dimethylamino)-1-propanol and the like, is reacted with about 1.0to about 1.3 equivalents of triphenylphosphine and about 1.0 to about1.3 equivalents of diethyl azodicarboxylate in an inert diluent, such astetrahydrofuran, at a temperature ranging from about −10° C. to about 5°C. for about 0.25 to about 1 hour. About 1.0 to about 1.3 equivalents ofa hydroxy compound, such as N-tert-butyltyrosine methyl ester, is thenadded and the reaction mixture is stirred at a temperature of about 0°C. to about 30° C. for about 2 to about 48 hours to provide theO-alkylated product.

In a similar manner, a compound of formula I, or an intermediatethereof, containing an aryl hydroxy group can be reacted with an aryliodide to provide a diaryl ether. Generally, this reaction is conductedby forming the alkali metal salt of the hydroxyl group using a suitablebase, such as sodium hydride, in an inert diluent, such as xylenes, at atemperature of about −25° C. to about 10° C. The salt is then treatedwith about 1.1 to about 1.5 equivalents of cuprous bromide dimethylsulfide complex at a temperature ranging from about 10° C. to about 30°C. for about 0.5 to about 2.0 hours, followed by about 1.1 to about 1.5equivalents of an aryl iodide, such as sodium 2-iodobenzoate and thelike. The reaction is then heated to a temperature from about 70° C. toabout 150° C. for about 2 to about 24 hours to provide the diaryl ether.

Additionally, a hydroxy-containing compound can also be readilyderivatized to form a carbamate. In one method for preparing suchcarbamates, a hydroxy compound of formula I, or an intermediate thereof,is contacted with about 1.0 to about 1.2 equivalents of 4-nitrophenylchloroformate in an inert diluent, such as dichloromethane, at atemperature ranging from about −25° C. to about 0° C. for about 0.5 toabout 2.0 hours. Treatment of the resulting carbonate. with an excess,preferably about 2 to about 5 equivalents, of a trialkylamine, such astriethylamine, for about 0.5 to 2 hours, followed by about 1.0 to about1.5 equivalents of a primary or secondary amine, provides the carbamate.Examples of amines suitable for using in this reaction include, but arenot limited to, piperazine, 1-methylpiperazine, 1-acetylpiperazine,morpholine, thiomorpholine, pyrrolidine, piperidine and the like.

Alternatively, in another method for preparing carbamates, ahydroxy-containing compound is contacted with about 1.0 to about 1.5equivalents of a carbamyl chloride in an inert diluent, such asdichloromethane, at a temperature ranging from about 25° C. to about 70°C. for about 2 to about 72 hours. Typically, this reaction is conductedin the presence of a suitable base to scavenge the acid generated duringthe reaction. Suitable bases include, by way of example, tertiaryamines, such as triethylamine, diisopropylethylamine, N-methylmorpholineand the like. Additionally, at least one equivalent (based on thehydroxy compound) of 4-(N,N-dimethylamino)pyridine is preferably addedto the reaction mixture to facilitate the reaction. Examples of carbamylchlorides suitable for use in this reaction include, by way of example,dimethylcarbamyl chloride, diethylcarbamyl chloride and the like.

Likewise, when a compound of formula I, or an intermediate thereof,contains a primary or secondary hydroxyl group, such hydroxyl groups canbe readily converted into a leaving group and displaced to form, forexample, amines, sulfides and fluorides. For example, derivatives of4-hydroxy-L-proline can be converted into the corresponding 4-amino,4-thio or 4-fluoro-L-proline derivatives via nucleophilic displacementof the derivatized hydroxyl group. Generally, when a chiral compound isemployed in these reactions, the stereochemistry at the carbon atomattached to the derivatized hydroxyl group is typically inverted.

These reactions are typically conducted by first converting the hydroxylgroup into a leaving group, such as a tosylate, by treatment of thehydroxy compound with at least one equivalent of a sulfonyl halide, suchas p-toluenesulfonyl chloride and the like, in pyridine. This reactionis generally conducted at a temperature of from about 0° C. to about 70°C. for about 1 to about 48 hours. The resulting tosylate can then bereadily displaced with sodium azide, for example, by contacting thetosylate with at least one equivalent of sodium azide in an inertdiluent, such as a mixture of N,N-dimethylformamide and water, at atemperature ranging from about 0° C. to about 37° C. for about 1 toabout 12 hours to provide the corresponding azido compound. The azidogroup can then be reduced by, for example, hydrogenation using apalladium on carbon catalyst to provide the amino (—NH₂) compound.

Similarly, a tosylate group can be readily displaced by a thiol to forma sulfide. This reaction is typically conducted by contacting thetosylate with at least one equivalent of a thiol, such as thiophenol, inthe presence of a suitable base, such as1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), in an inert diluent, such asN,N-dimethylformamide, at a temperature of from about 0° C. to about 37°C. for about 1 to about 12 hours to provide the sulfide. Additionally,treatment of a tosylate with morpholinosulfur trifluoride in an inertdiluent, such as dichloromethane, at a temperature ranging from about 0°C. to about 37° C. for about 12 to about 24 hours affords thecorresponding fluoro compound.

Furthermore, a compound of formula I, or an intermediate thereof, havinga substituent containing an iodoaryl group, for example, when R¹ is a4-iodophenyl group, can be readily converted either before or after theabove coupling reactions into a biaryl compound. Typically, thisreaction is conducted by treating the iodoaryl compound with about 1.1to about 2 equivalents of an arylzinc iodide, such as2-(methoxycarbonyl)phenylzinc iodide, in the presence of a palladiumcatalyst, such as palladium tetra(triphenylphosphine), in an inertdiluent, such as tetrahydrofuran, at a temperature ranging from about24° C. to about 30° C. until the reaction is complete. This reaction isfurther described, for example, in Rieke, J. Org. Chem. 1991, 56, 1445.

In some cases, the compounds of formula I, or intermediates thereof, maycontain substituents having one or more sulfur atoms. Such sulfur atomswill be present, for example, when the amino acid of formula II employedin the above reactions is derived from L-thiazolidine-4-carboxylic acid,L-(5,5-dimethyl)thiazolidine-4-carboxylic acid,L-thiamorpholine-3-carboxylic acid and the like. When present, suchsulfur atoms can be oxidized either before or after the above couplingreactions to provide a sulfoxide or sulfone compound using conventionalreagents and reaction conditions. Suitable reagents for oxidizing asulfide compound to a sulfoxide include, by way of example, hydrogenperoxide, 3-chloroperoxybenzoic acid (MCPBA), sodium periodate and thelike. The oxidation reaction is typically conducted by contacting thesulfide compound with about 0.95 to about 1.1 equivalents of theoxidizing reagent in an inert diluent, such as dichloromethane, at atemperature ranging from about −50° C. to about 75° C. for about 1 toabout 24 hours. The resulting sulfoxide can then be further oxidized tothe corresponding sulfone by contacting the sulfoxide with at least oneadditional equivalent of an oxidizing reagent, such as hydrogenperoxide, MCPBA, potassium permanganate and the like. Alternatively, thesulfone can be prepared directly by contacting the sulfide with at leasttwo equivalents, and preferably an excess, of the oxidizing reagent.Such reactions are described further in March, “Advanced OrganicChemistry”, 4th Ed., pp. 1201-1202, Wiley Publisher (1992).

Lastly, the compounds of formula I, where Q is —C(S)NR⁷—, can beprepared by using an amino thionoacid derivative in place of amino acidII in the above described synthetic procedures. Such amino thionoacidderivatives can be prepared by the procedures described in Shalaky, etal., J. Org. Chem., 61:9045-9048 (1996) and Brain, et al., J. Org.Chem., 62:3808-3809 (1997) and references cited therein.

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of formula I and IA areusually administered in the form of pharmaceutical compositions. Thesecompounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. These compounds are effective as both injectable and oralcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of formula I orIA above associated with pharmaceutically acceptable carriers. In makingthe compositions of this invention, the active ingredient is usuallymixed with an excipient, diluted by an excipient or enclosed within sucha carrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions suitably flavored with syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as. well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably, the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be breathed directly from thenebulizing device or the nebulizing device may be attached to a facemask tent or intermittent positive pressure breathing machine. Solution,suspension, or powder compositions may be administered, preferablyorally or nasally, from devices which deliver the formulation in anappropriate manner.

The following formulation examples illustrate the pharmaceuticalcompositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active mixture is mixed with the lactose and the mixture is added toa dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg  Starch 45.0mg  Microcrystalline cellulose 35.0 mg  Polyvinylpyrrolidone 4.0 mg (as10% solution in water) Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg  

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament, are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient, are made asfollows:

Ingredient Amount Active Ingredient   25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose, aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum  4.0 mg Sodiumcarboxymethyl cellulose (11%)/ 50.0 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to  5.0 ml

The medicament, sucrose and xanthan gum are blended, passed through aNo. 10 mesh U.S. sieve, and then mixed with,a previously made solutionof the microcrystalline cellulose and sodium carboxymethyl cellulose inwater. The sodium benzoate, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation Example 8

Quantity Ingredtent (mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 560 mg quantities.

Formulation Example 9

An intravenous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 250.0 mg Isotonic saline  1000 ml

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See. e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

It may be desirable or necessary to introduce the pharmaceuticalcomposition to the brain, either directly or indirectly. Directtechniques usually involve placement of a drug delivery catheter intothe host's ventricular system to bypass the blood-brain barrier. Onesuch implantable delivery system used for the transport of biologicalfactors to specific anatomical regions of the body is described in U.S.Pat. No. 5,011,472, which is herein incorporated by reference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Utility

The compounds of this invention can be employed to bind VLA-4 (α₄β₁integrin) in biological samples and, accordingly, have utility in, forexample, assaying such samples for VLA-4. In such assays, the compoundscan be bound to a solid support and the VLA-4 sample added thereto. Theamount of VLA-4 in the sample can be determined by conventional methodssuch as use of a sandwich ELISA assay. Alternatively, labeled VLA-4 canbe used in a competitive assay to measure for the presence of VLA-4 inthe sample. Other suitable assays are well known in the art.

In addition, certain of the compounds of this invention inhibit, invivo, adhesion of leukocytes to endothelial cells mediated by VLA-4 and,accordingly, can be used in the treatment of diseases mediated by VLA-4.Such diseases include inflammatory diseases in mammalian patients suchas asthma, Alzheimer's disease, atherosclerosis, AIDS dementia, diabetes(including acute juvenile onset diabetes), inflammatory bowel disease(including ulcerative colitis and Crohn's disease), multiple sclerosis,rheumatoid arthritis, tissue transplantation, tumor metastasis,meningitis, encephalitis, stroke, and other cerebral traumas, nephritis,retinitis, atopic dermatitis, psoriasis, myocardial ischemia and acuteleukocyte-mediated lung injury such as that which occurs in adultrespiratory distress syndrome.

The biological activity of the compounds identified above may be assayedin a variety of systems. For example, a compound can be immobilized on asolid surface and adhesion of cells expressing VLA-4 can be measured.Using such formats, large numbers of compounds can be screened. Cellssuitable for this assay include any leukocytes known to express VLA-4such as T cells, B cells, monocytes, eosinophils, and basophils. Anumber of leukocyte cell lines can also be used, examples include Jurkatand U937.

The test compounds can also be tested for the ability to competitivelyinhibit binding between VLA-4 and VCAM-1, or between VLA-4 and a labeledcompound known to bind VLA-4 such as a compound of this invention orantibodies to VLA-4. In these assays, the VCAM-1 can be immobilized on asolid surface. VCAM-1 may also be expressed as a recombinant fusionprotein having an Ig tail (e.g., IgG) so that binding to VLA-4 may bedetected in an immunoassay. Alternatively, VCAM-1 expressing cells, suchas activated endothelial cells or VCAM-1 transfected fibroblasts, can beused. For assays to measure the ability to block adhesion to brainendothelial cells, the assays described in International PatentApplication Publication No. WO 91/05038 are particularly preferred. Thisapplication is incorporated herein by reference in its entirety.

Many assay formats employ labelled assay components. The labellingsystems can be in a variety of forms. The label may be coupled directlyor indirectly to the desired component of the assay according to methodswell known in the art. A wide variety of labels may be used. Thecomponent may be labelled by any one of several methods. The most commonmethod of detection is the use of autoradiography with ³H, ¹²⁵S, ¹⁴C, or³²P labelled compounds or the like. Non-radioactive labels includeligands which bind to labelled antibodies, fluorophores,chemiluminescent agents, enzymes and antibodies which can serve asspecific binding pair members for a labelled ligand. The choice of labeldepends on sensitivity required, ease of conjugation with the compound,stability requirements, and available instrumentation.

Appropriate in vivo models for demonstrating efficacy in treatinginflammatory responses include EAE (experimental autoimmuneencephalomyelitis) in mice, rats, guinea pigs, or primates, as well asother inflammatory models dependent upon α₄ integrins.

Compounds having the desired biological activity may be modified asnecessary to provide desired properties such as improved pharmacologicalproperties (e.g., in vivo stability, bio-availability), or the abilityto be detected in diagnostic applications. For instance, inclusion ofone or more D-amino acids in the sulfonamides of this inventiontypically increases in vivo stability. Stability can be assayed in avariety of ways such as by measuring the half-life of the proteinsduring incubation with peptidases or human plasma or serum. A number ofsuch protein stability assays have been described (see, e.g., Verhoef,et al., Eur. J. Drug Metab. Pharmacokinet., 1990, 15(2):83-93).

For diagnostic purposes, a wide variety of labels may be linked to thecompounds, which may provide, directly or indirectly, a detectablesignal. Thus, the compounds of the subject invention may be modified ina variety of ways for a variety of end purposes while still retainingbiological activity. In addition, various reactive sites may beintroduced at the terminus for linking to particles, solid substrates,macromolecules, or the like.

Labeled compounds can be used in a variety of in vivo or in vitroapplications. A wide variety of labels may be employed, such asradionuclides (e.g., gamma-emitting radioisotopes such as technetium-99or indium-111), fluorescers (e.g., fluorescein), enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chemiluminescentcompounds, bioluminescent compounds, and the like. Those of ordinaryskill in the art will know of other suitable labels for binding to thecomplexes, or will be able to ascertain such using routineexperimentation. The binding of these labels is achieved using standardtechniques common to those of ordinary skill in the art.

In vitro uses include diagnostic applications such as monitoringinflammatory responses by detecting the presence of leukocytesexpressing VLA-4. The compounds of this invention can also be used forisolating or labeling such cells. In addition, as mentioned above, thecompounds of the invention can be used to assay for potential inhibitorsof VLA-4/VCAM-1 interactions.

For in vivo diagnostic imaging to identify, e.g., sites of inflammation,radioisotopes are typically used in accordance with well knowntechniques. The radioisotopes may be bound to the peptide eitherdirectly or indirectly using intermediate functional groups. Forinstance, chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar moleculeshave been used to bind proteins to metallic ion radioisotopes.

The complexes can also be labeled with a paramagnetic isotope forpurposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) orelectron spin resonance (ESR), both of which are well known. In general,any conventional method for visualizing diagnostic imaging can be used.Usually, gamma- and positron-emitting radioisotopes are used for cameraimaging and paramagnetic isotopes are used for MRI. Thus, the compoundscan be used to monitor the course of amelioration of an inflammatoryresponse in an individual. By measuring the increase or decrease inlymphocytes expressing VLA-4, it is possible to determine whether aparticular therapeutic regimen aimed at ameliorating the disease iseffective.

The pharmaceutical compositions of the present invention can be used toblock or inhibit cellular adhesion associated with a number of diseasesand disorders. For instance, a number of inflammatory disorders areassociated with integrins or leukocytes. Treatable disorders include,e.g., transplantation rejection (e.g., allograft rejection), Alzheimer'sdisease, atherosclerosis, AIDS dementia, diabetes (including acutejuvenile onset diabetes), retinitis, cancer metastases, rheumatoidarthritis, acute leukocyte-mediated lung injury (e.g., adult respiratorydistress syndrome), asthma, nephritis, and acute and chronicinflammation, including atopic dermatitis, psoriasis, myocardialischemia, and inflammatory bowel disease (including Crohn's disease andulcerative colitis). In preferred embodiments, the pharmaceuticalcompositions are used to treat inflammatory brain disorders, such asmultiple sclerosis (MS), viral meningitis and encephalitis.

Inflammatory bowel disease is a collective term for two similar diseasesreferred to as Crohn's disease and ulcerative colitis. Crohn's diseaseis an idiopathic, chronic ulceroconstrictive inflammatory diseasecharacterized by sharply delimited and typically transmural involvementof all layers of the bowel wall by a granulomatous inflammatoryreaction. Any segment of the gastrointestinal tract, from the mouth tothe anus, may be involved, although the disease most commonly affectsthe terminal ileum and/or colon. Ulcerative colitis is an inflammatoryresponse limited largely to the colonic mucosa and submucosa.Lymphocytes and macrophages are numerous in lesions of inflammatorybowel disease and may contribute to inflammatory injury.

Asthma is a disease characterized by increased responsiveness of thetracheobronchial tree to various stimuli potentiating paroxysmalconstriction of the bronchial airways. The stimuli cause release ofvarious mediators of inflammation from IgE-coated mast cells, includinghistamine, eosinophilic and neutrophilic chemotactic factors,leukotrines, prostaglandin and platelet activating factor. Release ofthese factors recruits basophils, eosinophils and neutrophils, whichcause inflammatory injury.

Atherosclerosis is a disease of arteries (e.g., coronary, carotid, aortaand iliac). The basic lesion, the atheroma, consists of a raised focalplaque within the intima, having a core of lipid and a covering fibrouscap. Atheromas compromise arterial blood flow and weaken affectedarteries. Myocardial and cerebral infarcts are a major consequence ofthis disease. Macrophages and leukocytes are recruited to atheromas andcontribute to inflammatory injury.

Rheumatoid arthritis is a chronic, relapsing inflammatory disease thatprimarily causes impairment and destruction of joints. Rheumatoidarthritis usually first affects the small joints of the hands and feetbut then may involve the wrists, elbows, ankles and knees. The arthritisresults from interaction of synovial cells with leukocytes thatinfiltrate from the circulation into the synovial lining of the joints.See e.g., Paul, Immunology, 3d ed., Raven Press (1993).

Another indication for the compounds of this invention is in treatmentof organ or graft rejection mediated by VLA-4. Over recent years therehas been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmunotolerance in the recipient to the transplanted allograft or organ.When allogeneic cells or organs are transplanted into a host (i.e., thedonor and donee are different individuals from the same species), thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue. CD8⁺ cells, CD4 cells andmonocytes are all involved in the rejection of transplant tissues.Compounds of this invention which bind to alpha-4 integrin are useful,inter alia, to block alloantigen-induced immune responses in th& donee,thereby preventing such cells from participating in the destruction ofthe transplanted tissue or organ. See, e.g., Paul et al., TransplantInternational 9: 420-425 (1996); Georczynski et al., Immunology 87:573-580 (1996); Georcyznski et al., Transplant. Immunol. 3: 55-61(1995); Yang et al., Transplantation 60: 71-76 (1996); Anderson et al.,APMIS 102: 23-27 (1994).

A related use for compounds of this invention which bind to VLA-4 is inmodulating the immune response involved in “graft versus host” disease(GVHD). See e.g., Schlegel et al., J. Immunol. 155: 3856-3865 (1995).GVHD is a potentially fatal disease that occurs when immunologicallycompetent cells are transferred to an allogeneic recipient. In thissituation, the donor's immunocompetent cells may attack tissues in therecipient. Tissues of the skin, gut epithelia and liver are frequenttargets and may be destroyed during the course of GVHD. The diseasepresents an especially severe problem when immune tissue is beingtransplanted, such as in bone marrow transplantation; but less severeGVHD has also been reported in other cases as well, including heart andliver transplants. The therapeutic agents of the present invention areused, inter alia, to block activation of the donor T-cells, therebyinterfering with their ability to lyse target cells in the host.

A further use of the compounds of this invention is inhibiting tumormetastasis. Several tumor cells have been reported to express VLA-4 andcompounds which bind VLA-4 block adhesion of such cells to endothelialcells. Steinback et al., Urol. Res. 23: 175-83 (1995); Orosz et al.,Int. J. Cancer 60: 867-71 (1995); Freedman et al., Leuk. Lymphoma 13:47-52 (1994); Okahara et al., Cancer Res. 54: 3233-6 (1994).

A further use of the compounds of this invention is in treating multiplesclerosis. Multiple sclerosis is a progressive neurological autoimmunedisease that affects an estimated 250,000 to 350,000 people in theUnited States. Multiple sclerosis is thought to be the result of aspecific autoimmune reaction in which certain leukocytes attack andinitiate the destruction of myelin, the insulating sheath covering nervefibers. In an animal model for multiple sclerosis, murine monoclonalantibodies directed against VLA-4 have been shown to block the adhesionof leukocytes to the endothelium, and thus prevent inflammation of thecentral nervous system and subsequent paralysis in the animals¹⁶.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985).

In order to enhance serum half-life, the compounds may be encapsulated,introduced into the lumen of liposomes, prepared as a colloid, or otherconventional techniques may be employed which provide an extended serumhalf-life of the compounds. A variety of methods are available forpreparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat.Nos. 4,235,871, 4,501,728 and 4,837,028, each of which is incorporatedherein by reference.

The amount administered to the patient will vary depending upon what isbeing administered, the purpose of the administration, such asprophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from a disease in anamount sufficient to cure or at least partially arrest the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend on the disease condition being treated as well asby the judgment of the attending clinician depending upon factors suchas the severity of the inflammation, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient are in the form ofpharmaceutical compositions described above. These compositions may besterilized by conventional sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention willvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. For example, for intravenous administration, the dose willtypically be in the range of about 20 μg to about 500 μg per kilogrambody weight, preferably about 100 μg to about 300 μg per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.1 pg to 1 mg per kilogram body weight. Effective dosescan be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. Unless otherwise stated, alltemperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

aq or aq. = aqueous AcOH = acetic acid bd = broad doublet bm = broadmultiplet bs = broad singlet Bn = benzyl Boc = N-tert-butoxylcarbonylBoc₂O = di-tert-butyl dicarbonate BOP =benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphateCbz = carbobenzyloxy CHCl₃ = chloroform CH₂Cl₂ ₌ dichloromethane (COCl)₂= oxalyl chloride d = doublet dd = doublet of doublets dt = doublet oftriplets DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene DCC =1,3-dicyclohexylcarbodiimide DMAP = 4-N,N-dimethylaminopyridine DME =ethylene glycol dimethyl ether DMF = N,N-dimethylformamide DMSO =dimethylsulfoxide EDC = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride Et₃N = triethylamine Et₂O = diethyl ether EtOAc = ethylacetate EtOH = ethanol eq or eq. = equivalent Fmoc =N-(9-fluorenylmethoxycarbonyl) FmocONSu =N-(9-fluorenylmethoxycarbonyl)-succinimide g = grams h = hour H₂O =water HBr = hydrobromic acid HCl = hydrochloric acid HOBT =1-hydroxybenzotriazole hydrate hr = hour K₂CO₃ = potassium carbonate L =liter m = multiplet MeOH = methanol mg = milligram MgSO₄ = magnesiumsulfate mL = milliliter mm = millimeter mM = millimolar mmol = millimolmp = melting point N = normal NaCl = sodium chloride Na₂CO₃ = sodiumcarbonate NaHCO₃ = sodium bicarbonate NaOEt = sodium ethoxide NaOH =sodium hydroxide NH₄Cl = ammonium chloride NMM = N-methylmorpholine Phe= L-phenylalanine Pro = L-proline psi = pounds per square inch PtO₂ =platinum oxide q = quartet q.s. = quantity sufficient quint. = quintetrt = room temperature s = singlet sat = saturated t = triplet t-BuOH =tert-butanol TFA = trifluoroacetic acid THF = tetrahydrofuran TLC or tlc= thin layer chromatography Ts = tosyl TsCl = tosyl chloride TsOH =tosylate μL = microliter

In the examples below, all temperatures are in degrees Celcius (unlessotherwise indicated). The following Methods were used to prepare thecompounds set forth below as indicated.

Method 1 N-Tosylation Procedure

N-Tosylation of the appropriate amino acid was conducted via the methodof Cupps, Boutin and Rapoport, J. Org. Chem. 1985, 50: 3972.

Method 2 Methyl Ester Preparation Procedure

Amino acid methyl esters were prepared using the method of Brenner andHuber, Helv. Chim. Acta, 1953, 36: 1109.

Method 3 BOP Coupling Procedure

The desired dipeptide ester was prepared by the reaction of a suitableN-protected amino acid (1 equivalent) with the appropriate amino acidester or amino acid ester hydrochloride (1 equivalent),benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate[BOP] (2.0 equivalent), triethylamine (1.1 equivalent), and DMF. Thereaction mixture was stirred at room temperature overnight. The crudeproduct is purified by flash chromatography to afford the dipeptideester.

Method 4 Hydrogenation Procedure I

Hydrogenation was performed using 10% palladium on carbon (10% byweight) in methanol at 30 psi overnight. The mixture was filteredthrough a pad of Celite and the filtrate concentrated to yield thedesired amino compound.

Method 5 Hydrolysis Procedure I

To a chilled (0° C.) THF/H₂O solution (2:1, 5-10 mL) of the appropriateester was added LiOH (or NaOH) (0.95 equivalents). The temperature wasmaintained at 0° C. and the reaction was complete in 1-3 hours. Thereaction mixture was extracted with ethyl acetate and the aqueous phasewas lyophilized resulting in the desired carboxylate salt.

Method 6 Ester Hydrolysis Procedure II

To a chilled (0° C.) THF/H₂O solution (2:1, 5-10 mL) of the appropriateester was added LiOH (1.1 equivalents). The temperature was maintainedat 0° C. and the reaction was complete in 1-3 hours. The reactionmixture was concentrated and the residue was taken up into H₂O and thepH adjusted to 2-3 with aqueous HCl. The product was extracted withethyl acetate and the combined organic phase was washed with brine,dried over MgSO₄, filtered and concentrated to yield the desired acid.

Method 7 Ester Hydrolysis Procedure III

The appropriate ester was dissolved in dioxane/H₂O (1:1) and 0.9equivalents of 0.5 N NaOH was added. The reaction was stirred for 3-16hours and then concentrated. The resulting residue was dissolved in H₂Oand extracted with ethyl acetate. The aqueous phase was lyophilized toyield the desired carboxylate sodium salt.

Method 8 Sulfonylation Procedure I

To the appropriately protected aminophenylalanine analog (11.2 mmol),dissolved in methylene chloride (25 ml) and cooled to −78° C., was addedthe desired sulfonyl chloride (12 mmol) followed by dropwise addition ofpyridine (2 mL). The solution was allowed to warm to room temperatureand was stirred for 48 hr. The reaction solution was transferred to a250 mL separatory funnel with methylene chloride (100 mL) and extractedwith 1N HCl (50 mL×3), brine (50 mL), and water (100 mL). The organicphase was dried (MgSO⁴) and the solvent concentrated to yield thedesired product.

Method 9 Reductive Amination Procedure

Reductive amination of Tos-Pro-p-NH²-Phe with the appropriate aldehydewas conducted using acetic acid, sodium triacetoxyborohydride andmethylene chloride, and the combined mixture was stirred at roomtemperature overnight. The crude product was purified by flashchromatography.

Method 10 BOC Removal Procedure

Anhydrous hydrochloride (HCl) gas was bubbled through a methanolicsolution of the appropriate Boc-amino acid ester at 0° C. for 15 minutesand the reaction mixture was stirred for three hours. The solution wasconcentrated to a syrup and dissolved in Et₂O and reconcentrated. Thisprocedure was repeated and the resulting solid was placed under highvacuum overnight.

Method 11 Tert-Butyl Ester Hydrolysis Procedure I

The tert-butyl ester was dissolved in CH₂Cl₂ and treated with TFA. Thereaction was complete in 1-3 hr, at which time the reaction mixture wasconcentrated and the residue dissolved in H₂O and lyophilized to yieldthe desired acid.

Method 12 EDC Coupling Procedure I

To a CH₂Cl₂ solution (5-20 mL) of N-(toluene-4-sulfonyl)-L-proline (1equivalent), the appropriate amino acid ester hydrochloride (1equivalent), N-methylmorpholine (1.1-2.2 equivalents) and1-hydroxybenzotriazole (2 equivalents) were added and mixed, placed inan ice bath and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (1.1equivalents) added. The reaction was allowed to rise to room temperatureand was stirred overnight. The reaction mixture was poured into H₂O andthe organic phase was washed with sat. NaHCO₃ and brine, dried (MgSO₄ orNa₂SO₄), filtered and concentrated. The crude product was purified bycolumn chromatography.

Method 13 EDC Coupling Procedure II

To a DMF solution (5-20 mL) of the appropriate N-protected amino acid (1equivalent), the appropriate amino acid ester hydrochloride (1equivalent), Et₃N (1.1 equivalents) and 1-hydroxybenzotriazole (2equivalents) were added and mixed, placed in an ice bath and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (1.1 equivalents) added.The reaction was allowed to rise to room temperature and was stirredovernight. The reaction mixture was partitioned between EtOAc and H₂Oand the organic phase washed with 0.2 N citric acid, H₂O, sat. NaHCO₃and brine, dried (MgSO₄ or Na₂SO₄), filtered and concentrated. The crudeproduct was purified by column chromatography or preparative TLC.

Method 14 Sulfonylation Procedure II

The appropriate sulfonyl chloride was dissolved in CH₂Cl₂ and placed inan ice bath. L-Pro-L-Phe-OMe.HCl (1 equivalent) and Et₃N (1.1equivalent) was added and the reaction allowed to warm to roomtemperature and stirred overnight under an atmosphere of nitrogen. Thereaction mixture was concentrated and the residue partitioned betweenEtOAc and H₂O, and the organic phase was washed with sat. NaHCO₃ andbrine, dried (MgSO₄ or Na₂SO₄), filtered and concentrated. The crudeproduct was purified by column chromatography or preparative TLC.

Method 15 Sulfonylation Procedure III

To a solution of L-Pro-L-4-(3-dimethylaminopropyloxy)-Phe-OMe (1equivalent) [prepared using the procedure described in Method 10] inCH₂Cl₂ was added Et₃N (5 equivalents) followed-by the appropriatesulfonyl chloride (1.1 equivalent). The reaction was allowed to warm toroom temperature and stirred overnight under an atmosphere of nitrogen.The mixture was concentrated, dissolved in EtOAc, washed with sat.NaHCO₃ and 0.2 N citric acid. The aqueous phase was made basic withsolid NaHCO₃ and the product extracted with EtOAc. The organic phase waswashed with brine, dried (MgSO₄ or Na₂SO₄), filtered and concentrated.The crude methyl ester was purified by preparative TLC. Thecorresponding acid was prepared using the procedure described in Method7.

Method 16 Hydrogenation Procedure II

To a methanol (10-15 mL) solution of the azlactone was added NaOAc (1equivalent) and 10% Pd/C. This mixture was placed on the hydrogenator at40 psi H₂. After 8-16 hours, the reaction mixture was filtered through apad of Celite and the filtrate concentrated to yield thedehydrodipeptide methyl ester. The ester was dissolved in dioxane/H₂O(5-10 mL), to which was added 0.5 N NaOH (1.05 equivalents). Afterstirring for 1-3 hours, the reaction mix was concentrated and theresidue was redissolved in H₂O and washed with EtOAc. The aqueous phasewas made acidic with 0.2 N HCl and the product was extracted with EtOAc.The combined organic phase was washed with brine (1×5 mL,), dried (MgSO₄or Na₂SO₄), filtered and concentrated to yield the acid as approximatelyand mixture of diastereomers.

Method 17 Tert-Butyl Ester Hydrolysis Procedure II

The tert-butyl ester was dissolved in CH₂Cl₂ (5 mL) and treated with TFA(5 mL). The reaction was complete in 1-3 hours, at which time thereaction mixture was concentrated and the residue dissolved in H₂O andconcentrated. The residue was redissolved in H₂O and lyophilized toyield the desired product.

Example 1 Synthesis of N-(Methanesulfonyl)-L-prolyl-L-phenylalanine

Boc-L-Pro-OH and L-Phe-OBn.Hcl were treated with BOP and NMM in DMF togive, after aqueous workup and flash chromatography,Boc-L-Pro-L-Phe-OBn. This product was then treated with TFA and anisole,and the mixture was evaporated. The residue was dissolved in Et₂O andwashed with saturated aqueuos NaHCO₃ and saturated aqueous NaCl. TheEt₂O layer was dried over anhydrous MgSO₄, filtered, and the solventevaporated to give L-Pro-L-Phe-OBn. The residue was treated withCH₃SO₂Cl and Et₃N in CH₂Cl₂ to give, after aqueous workup and flashchromatography, N-(CH₃SO₂)-L-Pro-L-Phe-OBn. This product was thentreated with 10% Pd on C in THF, and the mixture was shaken under 50 psiH₂. The mixture was filtered through Celite, and evaporated to give thetitle compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=7.93 (d, J=8.2,1H), 7.27-7.13 (m, 5H), 4.51-4.45 (m, 1H), 4.15 (dd, J=8.7, J=2.6, 1H),3.59 (t, J=6.1, 1H), 3.30-3.25 (m, 2H), 3.10 (dd, J=13.7, J=4.9, 1H),2.96 (dd, J=13.7, J=9.1, 1H), 2.87 (s, 3H), 2.03-1.93 (m, 1H), 1.77-1.54(m, 3H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.6, 171.1, 137.4, 129.2, 128.1,126.4, 61.2, 53.0, 48.6, 36.5, 35.2, 30.6, 24.0. Mass Spectroscopy:(+FAB, 3-nitrobenzyl alcohol) 341 (MH+).

Example 2 Synthesis of N-(α-Toluenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of PhCH₂SO₂Cl for CH₃SO₂Cl, and following the methods forpreparation of Example 1 (9), gave the title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.01 (d, J=8.2,1H), 7.39-7.20 (m, 5H), 7.18-7.13 (m, 5H), 4.53-4.46 (m, 1H), 4.42 (d,J=13.5, 1H), 4.35 (d, J=13.5, 1H), 4.20-4.17 (m, 1H), 3.31-3.18 (m, 3H),3.09 (dd, J=13.9, J=4.8, 1H), 2.96 (dd, J=13.8, J=8.9, 1H), 2.03-1.95(m, 1H), 1.79-1.58 (m, 3H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.6, 171.5,137.6, 131.0, 129.3, 129.0, 128.9, 128.1, 128.0, 126.3, 61.5, 53.3,48.8, 36.5, 30.8, 30.6, 24.0. Mass Spectroscopy: (+FAB, 3-nitrobenzylalcohol) 417 (MH+).

Example 3 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanine

Substitution of 4-CH₃(C₆H₄)SO₂Cl for CH₃SO₂Cl, and following the methodsfor preparation of Example 1 (9), gave the title compound as a clearoil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.07 (d, J=8.0,1H), 7.68 (d, J=8.2, 2H), 7.39 (d, J=8.1, 2H), 7.29-7.15 (m, 5H),4.524.44 (m, 1H), 4.10-4.07 (m, 1H), 3.34-3.27 (m, 1H), 3.12-3.06 (m,2H), 2.98 (dd, J=13.7, J=8.7, 1H), 2.39 (s, 3H), 1.57-1.36 (m, 4H). ¹³CNMR (DMSO-d₆, 75 MHz): δ=172.6, 170.8, 143.6, 137.4, 133.8, 129.8,129.3, 128.1, 127.5, 126.4, 61.3, 53.2, 49.0, 36.6, 30.4, 23.7, 21.0.Mass Spectroscopy: (+FAB, glycerol/trifluoroacetic acid) 417 (MH+).

Example 4 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-L-(N-methyl)phenylalanine

N-Methyl-L-Phe-OH was treated with benzyl alcohol and4-methylphenylsulfonic acid to give N-Methyl-L-Phe-OBn TsOH.Substitution of L-Pro-OH for D-Pro-OH, and substitution ofN-Methyl-Phe-OBn.TsOH for L-Phe-OBn.TsOH, and following the methods forpreparation of Example 6 (17), gave the title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=7.60 (d, J=8.2,2H), 7.34 (d, J=8.2, 2H), 7.27-7.16 (m, 5H), 4.97-4.92 (m, 1H),4.64-4.61 (m, 1H), 3.33 (bs, 1H), 3.25-3.12 (m, 3H), 3.04 (dd, J=10.5,J=14.6, 1H), 2.85 (s, 3H), 2.37 (s, 3H), 1.86-1.72 (m, 2H), 1.68-1.50(m, 2H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=171.9, 171.1, 143.0, 137.9, 135.6,129.6, 128.7, 128.2, 127.2, 126.3, 58.6, 48.0, 33.4, 33.0, 30.4, 29.6,23.9, 21.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 431 (MH+).

Example 5 Synthesis ofN-(Toluene-4-sulfonyl)-L-pipecolinyl-L-phenylalanine

Substitution of L-pipecolinic acid for D-Pro-OH, and following themethods for preparation of Example 6 (17), gave the title compound as aclear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.09 (d, J=8.0,1H), 7.44 (d, J=8.2, 2H), 7.34-7.21 (m, 5H), 7.17 (d, J=8.2, 2H), 4.44(d, J=4.8, 1H), 4.324.24 (m, 1H), 3.61-3.53 (m, 2H), 3.33 (bs, 1H),3.17-3.11 (m, 1H), 3.07 (dd, J=13.8, J=4.5, 1H), 2.89 (dd, J=13.8,J=9.9, 1H), 2.33 (s, 3H), 1.88 (bd, J=12.9, 1H), 1.40-1.30 (m, 3H),1.08-1.05 (m, 2H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.8, 169.8, 142.7,137.7, 136.6, 129.4, 129.2, 128.2, 126.8, 126.5, 54.1, 53.4, 42.4, 36.3,30.4, 26.7, 23.5, 21.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol)431 (MH+).

Example 6 Synthesis of N-(Toluene-4-sulfonyl)-D-prolyl-L-phenylalanine

D-Pro-OH was treated with TsCl and NaOH in H₂O to give, afteracidification, extraction, drying over anhydrous MgSO₄, and evaporation,N-(Toluene-4-sulfonyl)-D-Pro-OH. This product was treated withH-Phe-OBn.Hcl, BOP, and NMM in DMF to give, after aqueous workup andflash chromatography, N-(toluene-4-sulfonyl)-D-Pro-Phe-OBn. This productwas treated with 10% Pd on C in THF, and the mixture was shaken under 50psi H₂. The mixture was filtered through celite and evaporated to givethe title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.10 (d, J=8.4,1H), 7.70 (d, J=8.2, 2H), 7.40 (d, J=8.2, 2H), 7.28-7.15 (m, 5H),4.51-4.44 (m, 1H), 4.13 (t, J=5.7, 1H), 3.33-3.25 (m, 2H), 3.11-3.03 (m,2H), 2.94 (dd, J=13.8, J=9.0, 1H), 2.39 (s, 3H), 1.66-1.57 (m, 1H),1.50-1.37 (m, 3H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.6, 170.8, 143.5,137.3, 134.1, 129.8, 129.3, 128.1, 127.4, 126.4, 61.3, 53.0, 48.9, 36.8,30.4, 23.8, 21.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 417(MH+).

Example 7 Synthesis ofN-(Toluene-4-sulfonyl)-L-(trans-4-hydroxy)-prolyl-L-phenylalanine

Substitution of trans-4-hydroxy-L-proline for D-Pro-OH, and followingthe methods for preparation of Example 6 (17); gave the title compoundas a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.25 (d, J=8.1,1H), 7.70 (d, J=8.2, 2H), 7.36 (d, J=8.2, 2H), 7.30-7.15 (m, 5H), 4.81(bs, 1H), 4.47-4.40 (m, 1H), 4.154.310 (m, 2H), 3.44 (dd, J=10.3, J=4.8,1H), 3.35 (bs, 1H), 3.09-2.91 (m, 3H), 2.38 (s, 3H), 1.72-1.64 (m, 2H).¹³C NMR (DMSO-d₆, 75 MHz): δ=172.6, 171.0, 143.1, 137.4, 134.1, 129.5,129.3, 128.1, 127.7, 126.4, 68.1, 60.3, 56.1, 53.4, 36.7, 30.4, 21.0.Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 433 (MH+).

Example 8 Synthesis ofN-(Toluene-4-sulfonyl)-L-(azetidine-2-carbonyl)-L-phenylalanine

Substitution of (S)-2-azetidinecarboxylic acid for D-Pro-OH, andfollowing the methods for preparation of Example 6 (17), gave the titlecompound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.12 (d, J=8.0,1H), 7.69 (d, J=8.2, 2H), 7.47 (d, J=8.2, 2H), 7.31-7.13 (m, 5H),4.54-4.47 (m, 1H), 4.23 (t, J=8.3, 1H), 3.67-3.58 (m, 2H), 3.47 (q,J=8.4, 1H), 3.33 (bs, 1H), 3.10 (dd, J=13.7, J=5.2, 1H), 2.98 (dd,J=13.6, J=7.9, 1H), 2.42 (s, 3H), 2.03-1.87 (m, 2H). ¹³C NMR (DMSO-d₆,75 MHz): δ=172.3, 168.7, 144.3, 137.1, 130.9, 130.0, 129.4, 128.3,128.2, 126.6, 61.8, 53.1, 47.9, 36.7, 21.1, 19.6. Mass Spectroscopy:(+FAB, 3-nitrobenzyl alcohol) 403 (MH+).

Example 9 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-D,L-homophenylalanine

N-(toluene-4-sulfonyl)-L-proline (250 mg, 0.92 mmol) was dissolved inDMF (20 mL) with D,L-homophenylalanine methyl ester (166 mg, 1.1 eq),Et₃N (2.1 eq, 283 μL), and BOP (1.1 eq, 410 mg). The dipeptide wasisolated in 73% yield (300 mg, 0.67 mmol) as an oil. The ester (300 mg,0.67 mmol) was then hydrolyzed in a 1:1 MeOH:H₂O (5 mL) solution withNaOH (1.1 eq, 28 mg). The acid was isolated as a foam in 60% yield (175mg, 0.40 mmol).

NMR data was as follows: ¹H NMR (300 MHz, CD₃OD): δ=7.60 (m, 2H), 7.20(m, 2H), 7.03 (m, 5H), 4.23-3.87 (m, 2H), 3.42 (m, 1H), 3.29 (m, 1H),2.58 (m, 2H), 2.20 (s, 3H), 2.02-1.38 (m, 7H). ¹³C NMR (75 MHz, CD₃OD):δ=175.53, 175.17, 146.49, 146.19, 142.93, 135.91, 135.04, 131.72,130.28, 129.61, 129.49, 129.21, 127.70, 64.39, 63.59, 53.76, 53.52,51.55, 51.17, 35.15, 33.42, 32.74, 32.60, 32.48, 26.28, 26.20, 22.14.Mass Spectroscopy: (FAB) 431 (M+H).

Example 10 Synthesis ofN-(4-Chlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of 4-chlorophenyl-SO₂Cl for CH₃SO₂Cl, and following themethods for the preparation of Example 1 (9), gave the title compound asa solid, mp=69-71° C.

NMR data was as follows: ¹H NMR (300 MHz, CDCl₃): δ=7.77 (m, 2H), 7.53(m, 2H), 7.31 (m, 5H), 4.72 (m, 1H), 4.04 (m, 1H), 3.35 (m, 2H), 3.10(m, 2H), 2.05 (m, 1H), 1.54 (m, 3H). ¹³C NMR (75 MHz, CD₃OD): δ=174.67,174.41, 141.23, 138.81, 137.55, 135.02, 131.18, 130.04, 129.45, 128.45,63.75, 55.48, 38.79, 32.32, 25.87 (1C buried under solvent peak). MassSpectroscopy: (FAB) 437 (M+H).

Example 11 Synthesis ofN-(1-Naphthalenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of 1-naphthyl-SO₂Cl for CH₃SO₂Cl, and following the methodsfor preparation of Example 1 (9), gave the title compound as a clearoil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.73-8.70 (m, 1H),8.24 (d, J=8.2, 1H), 8.12-8.06 (m, 2H), 7.69-7.57 (m, 2H), 7.33-7.13 (m,7H), 4.40-4.33 (m, 2H), 3.34-3.23 (m, 3H), 3.03 (dd, J=14.0, J=5.2, 1H),2.88 (dd, J=13.7, J=8.8, 1H), 1.79-1.15 (m, 4H). ¹³C NMR (DMSO-d₆, 75MHz): δ=172.6, 170.9, 137.4, 134.3, 134.0, 133.6, 129.2, 129.1, 129.0,128.23, 128.18, 128.1, 126.9, 126.4, 124.7, 124.6, 60.6, 53.4, 48.7,36.5, 30.8, 24.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 475(Mna+).

Example 12 Synthesis ofN-(2-Naphthalenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of 2-naphthyl-SO₂Cl for CH₃SO₂Cl, and following the methodsfor preparation of Example 1 (9), gave the title compound as a clearoil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.18-8.03 (m, 4H),7.82 (dd, J=8.7, J=1.8, 1H), 7.74-7.64 (m, 2H), 7.32-7.15 (m, 6H),4.534.46 (m, 1H), 4.25-4.21 (m, 1H), 3.34-3.27 (m, 3H), 3.10 (dd,J=13.8, J=5.1, 1H), 2.99 (dd, J=13.7, J=8.8, 1H), 1.56-1.36 (m, 4H). ¹³CNMR (DMSO-d₆, 75 MHz): δ=172.6, 170.8, 137.4, 134.4, 133.9, 131.8,129.4, 129.3, 129.0, 128.7, 128.1, 127.8, 127.6, 127.4, 122.8, 61.4,53.2, 49.1, 36.6, 30.4, 23.8. Mass Spectroscopy: (+FAB, 3-nitrobenzylalcohol) 475 (Mna+).

Example 13 Synthesis ofN-(4-Methoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of 4-methoxyphenyl-SO₂Cl for CH₃SO₂Cl, and following themethods for the preparation of Example 1 (9), gave the title compound asa solid.

NMR data was as follows: ¹H NMR (300 MHz, CDCl₃): δ=7.77 (m, 2H), 7.31(m, 5H), 7.01 (m, 2H), 4.80 (m, 1H), 4.05 (m, 1H), 3.86 (s, 3H), 3.35(m, 2H), 3.10 (m,2H), 1.96 (m, 1H), 1.54 (m, 3H). ¹³C NMR (75 MHz,CD₃OD): δ=174.69, 174.54, 165.63, 136.75, 131.69, 131.07, 130.02,128.45, 116.13, 63.84, 56.83, 55.35, 51.09, 38.81, 32.15, 26.63. MassSpectroscopy: (FAB) 433 (M+H).

Example 14 Synthesis ofN-(4-tert-Butylbenzenesulfonyl)-L-prolyl-L-phenylalanine

Substitution of 4-tert-butylphenyl-SO₂Cl for CH₃SO₂Cl, and following themethods for preparation of Example 1 (9), gave the title compound as asolid.

NMR data was as follows: ¹H NMR (300 MHz, CDCl₃): δ=7.75 (d, 2H, J=8.52Hz), 7.55 (d, 2H, J=8.43 Hz), 7.29 (m, 5H), 4.80 (m, 1H), 4.05 (m, 1H),3.30 (m, 3H), 3.10 (m, 3H), 1.95 (m, 1H), 1.56 (m, 2H), 1.34 (s, 9H).¹³C NMR (75 MHz, CD₃OD): δ=174.81, 174.47, 159.01, 138.79, 135.59,131.09, 130.02, 129.45, 128.43, 128.05, 63.83, 55.46, 51.09, 38.84,36.66, 32.20, 31.99, 25.83. Mass Spectroscopy: (FAB) 459 (M+H).

Example 15 Synthesis ofN-(Toluenesulfonyl)-L-(trans-4-fluoro)-prolyl-L-phenylalanine

The product from Example 49 (176) was treated with 10% Pd on C in THFand the mixture was shaken under 50 psi H₂. The mixture was filteredthrough celite and evaporated to give the title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.43 (d, J=8.1,1H), 7.66 (d, J=8.3, 2H), 7.35 (d, J=8.3, 2H), 7.29-7.13 (m, 5H), 5.10(bd, J=53.3, 1H), 4.49-4.42 (m, 1H), 4.19 (t, J=8.3, 1H), 3.63-3.46 (m,2H), 3.34 (bs, 1H), 3.06 (dd, J=13.7, J=5.4, 1H), 2.95 (dd, J=13.7,J=8.3, 1H), 2.38 (s, 3H), 2.25-2.12 (m, 1H), 1.94-1.72 (m, 1H). ¹³C NMR(DMSO-d₆, 75 MHz): δ=172.5, 170.4, 143.4, 137.4, 134.2, 129.5, 129.4,128.1, 127.6, 126.5, 92.0 (d, J=177 Hz), 59.9, 55.1, 53.4, 37.6 (d, J=21Hz), 36.8, 21.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 435(MH+).

Example 16 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-fluoro)-prolyl-L-phenylalanine

The product from Example 50 (177) was treated with 10% Pd on C in THFand was shaken under 50 psi H₂. The mixture was filtered through celiteand evaporated to give the title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=7.78 (d, J=7.7,1H), 7.73 (d, J=8.2, 2H), 7.41 (d, J=8.2, 2H), 7.29-7.13 (m, 5H), 5.10(dt, Jd=52.9, Jt=3.5, 1H), 4.53-4.46 (m, 1H), 4.31-4.27 (m, 1H),3.60-3.28 (m, 3H), 3.06-2.94 (m, 2H), 2.39 (s, 3H), 2.18 (dd, J=19.8,J=15.2, 1H), 1.93-1.70 (m, 1H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.1,169.7, 144.1, 136.8, 132.8, 130.0, 129.3, 128.1, 127.7, 126.6, 92.0 (d,J=176 Hz), 60.5, 55.1 (d, J=23.5), 53.2, 36.9, 36.2, 21.0. MassSpectroscopy: (+FAB, 3-nitrobenzyl alcohol) 435 (MH+).

Example 17 Synthesis ofN-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)-thiaprolyl-L-phenylalanine

N-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)-thiazolidine4-carboxylic acidwas prepared from L-(5,5-dimethyl)-thiazolidine-4-carboxylic acid usingthe procedure described in Method 1. The title compound was preparedaccording to the procedure described for Example 1 (9) to provide for asolid having a mp=79-81° C.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=0.98 (s, 3H), 1.08(s, 3H), 2.42 (s, 3H), 3.04-3.34 (m, 2H), 3.88 (s, 1H), 4.38 (d, 1H,J=10.3 Hz), 4.52 (d, 1H, J=10.2 Hz), 4.90 (m, 1H), 7.09 (bd, 1H),7.15-7.37 (m, 7H), 7.73 (d, 2H, J=8.2 Hz). ¹³C NMR (CDCl₃, 75 MHz):δ=22.3, 24.3, 29.6, 38.3, 51.2, 54.1, 55.3, 73.9, 127.7, 128.7, 129.2,130.1, 130.7, 133.0, 136.5, 145.6, 169.9, 174.5. Mass Spectroscopy:(FAB+) 463 (M+H).

Example 18 Synthesis ofN-(2-Methoxycarbonylbenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared from the product of Example 53 using theprocedure described in Method 4, yielding a yellow solid, mp=163-165° C.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=8.74 (bs, 1H); 7.93(d, 1H, J=7.57 Hz); 7.70 (d, 1H, J=8.12 Hz); 7.57 & 7.25 (M, 8H); 4.69(m, 1H); 4.39 (d, 1H, J=7.69 Hz); 3.93 (s, 3H); 3.44 (m, 1H); 3.29 (m,2H); 2.93 (m, 1H); 1.88, 1.62 & 1.17 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz):δ=175.67, 172.45, 169.27, 137.19, 135.39, 134.12, 133.67, 131.42,130.55, 129.77, 129.52, 129.09, 127.52, 62.19, 54.08, 49.34, 37.58,31.48, 30.89, 24.21. Mass Spectroscopy: (+FAB) 461 (M+H) 483 (M+Na).

Example 19 Synthesis ofN-(2-Carboxybenzenesulfonyl)-L-prolyl-L-phenylalanine

The product of Example 18 (56) (1.8 mmoles) was mixed with dioxane (25mL) and 1N NaOH (1.8 mmoles) and the reaction was stirred at roomtemperature for 16 hours. 1N HCl (1.8 mmoles) was added and the waterand dioxane were removed under reduced pressure. The product wasextracted with 0.2 N NaOH (50 mL) and EtOAc (2×50 mL) and the recoveredorganic layer was dried over MgSO₄, filtered, and concentrated to yielda colorless oil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=11.15 (bs, 2H);7.95, 7.57, & 7.21 (m, 9H); 4.74 (m, 1H); 4.53 (m, 1H); 3.44 (m, 1H);3.28 (m, 2H); 2.91 (m, 1H); 1.89 (M, 2H); 1.60 (m, 1H); 1.06 (m, 1H).¹³C NMR (CDCl₃, 300 MHz): δ=175.75, 173.60, 171.31, 136.81, 135.18,133.72, 131.58, 130.81, 129.70, 129.18, 127.66, 67.52, 61.99, 53.95,49.36, 45.09, 37.40, 24.19, 21.66. Mass Spectroscopy: (+FAB) 447 (M+H).

Example 20 Synthesis ofN-(Toluene-4-sulfonyl)-L-thiaprolyl-L-phenylalanine

N-(Toluene-4-sulfonyl)-L-thiazolidine4-carboxylic acid was prepared fromL-thiazolidine-4-carboxylic acid using the procedure described inMethod 1. The title compound was prepared according to the proceduredescribed for Example 1 (9) as a solid, mp=60-65° C.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=2.43 (s, 5H),3.0-3.40 (m, 3H), 4.0 (d, 1H, J=15.7 Hz), 4.55 (d, 1H, J=15.8 Hz), 4.64(m, 1H), 4.87 (m, 1H), 7.19-7.41 (m, 7H), 7.72 (m, 2H). ¹³C NMR (CDCl₃,75 MHz): δ=22.3, 33.6, 37.7, 52.0, 53.0, 65.8, 127.8, 1-28.5, 129.4,129.9, 130.7, 133.9, 136.2, 145.7, 169.4, 175.0. Mass Spectroscopy:(FAB+) 435 (M+H).

Example 21 Synthesis ofN-(3,5-Dichlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

Proline methyl ester hydrochloride (2.68 g, 16.2 mmol) was dissolved inpyridine (20 mL) and 3,5-dichlorophenylsulfonyl chloride (3.57 g, 14.6mmol) was added to the mixture and stirred for 19 hr. Water (5 mL) wasadded and the mixture was stirred for 45 minutes before diluting withwater (200 mL). The mixture was extracted with Et₂O (2×150 mL) and thecombined extracts were washed with water (3×100 mL), 1N HCl (150 mL) andsaturated aqueous NaHCO₃ (150 mL), then dried (MgSO₄), filtered andevaporated in vacuo to give 3,5-dichlorophenylsulfonylproline methylester as an oil (4.34 g, 88%). The methyl ester was dissolved in MeOH(20 mL) and 1N NaOH (20 mL) was added. The mixture was gently warmeduntil homogeneous and then stirred for 1.5 hr. The solvent was removedin vacuo and the residue taken up in water (50 mL). The aqueous solutionwas washed with Et₂O (30 mL) and then made acidic with 12 N HCl. Themixture was extracted with CHCl₃ (2×40 mL) and the combined extractswere dried over anhydrous MgSO₄, filtered and the solvent evaporated invacuo to give 3,5-dichlorophenylsulfonyl-L-proline as a solid (3.37 g,83%). 3,5-Dichlorophenylsulfonyl-L-proline was coupled toL-phenylalanine ethyl ester using the procedure described in Method 3above to give the corresponding dipeptide ethyl ester (348 mg, 45%). Thetitle compound was prepared via hydrolysis of the ethyl ester using NaOHin MeOH (61 mg, 19%). NMR analysis indicated that diastereomers werepresent.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.11 (bs, 1H), 7.72 (d, 2H,J=1.9 Hz), 7.61 (q, 1H, J=2.0 Hz), 7.39-7.20 (6H), 4.92 (m, 1H), 4.15(m, 1H); 3.52-3.09 (4H), 2.11 (m, 1H), 1.95-1.49 (3H). ¹³C NMR (CDCl₃):δ=175.4, 175.2, 171.8, 171.7, 139.5, 139.3, 137.0, 136.6, 135.8, 134.0,134.0, 129.9, 129.5, 127.7, 126.7, 111.4, 63.1, 62.9, 53.8, 53.6, 50.5,50.3, 45.1, 37.9, 31.3, 30.8, 24.8, 24.6. Mass Spectroscopy: FAB m/e 471(M+H).

Example 22 Synthesis ofN-(4-Trifluoromethoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as described in Example 21 (59) except4-trifluoromethoxyphenylsulfonyl chloride was used in place of3,5-dichlorophenylsulfonyl chloride.

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.90 (d, 2H, J=8.8 Hz),7.38-7.21 (8H), 6.90 (bs, 1H), 4.90 (m, 1H), 4.15 (dd, 1H, J=2.5, 8.4Hz), 3.38 (m, 2H), 3.12 (m, 2H), 1.98 (m, 1H), 1.55 (2H), 1.41 (m, 1H).¹³C NMR (CDCl₃): δ=174.9, 172.0, 153.3, 136.6, 134.9, 130.6, 129.9,129.2, 127.7, 121.7, 62.8, 53.8, 50.2, 37.9, 30.6,-24.6. MassSpectroscopy: FAB m/e 487 (M+H).

Example 23 Synthesis ofN-(3,4-Dichlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as in Example 21 (59) except3,4-dichlorophenylsulfonyl chloride was used in place of3,5-dichlorophenylsulfonyl chloride.

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.94 (d, 1H, J=1.9 Hz), 7.64(m, 2H), 7.55 (bs, 1H), 7.36-7.21 (6H), 4.92 (dt, 1H, J=5.2, 8.0 Hz),4.16 (m, 1H), 3.36 (m, 2H), 3.11 (m, 1H), 1.98 (m, 1H), 1.58 (m, 2H),1.40 (m, 1H). ¹³C NMR (CDCl₃): δ=175.0, 171.8, 139.5, 136.4, 134.7,132.1, 130.2, 129.9, 129.2, 127.7, 127.4, 62.9, 53.8, 50.3, 37.9, 30.7,24.6. Mass Spectroscopy: FAB m/e 471 (M+H).

Example 24 Synthesis ofN-(Toluene-4-sulfonyl)-D,L-(trans-3-phenyl)prolyl-L-phenylalanline

N-(Toluene-4-sulfonyl)-trans-3-phenylproline (prepared via the method ofChung et al., J. Org. Chem., 55: 270-275 (1990)) was coupled toL-phenylalanine ethyl ester using the procedure described in Method 3and purified by silica gel flash chromatography (93/7 CH₂Cl₂/MeOH) togive the ethyl ester of the title compound as a white solid. The acidwas prepared via hydrolysis of the ethyl ester using NaOH in ethanol.

NMR data was as follows: ¹H NMR (DMSO-d₆ d 8.38 (m, 1H), 7.70 (m, 2H),7.40-7.06 (10H), 6.80 (m, 1H), 6.62 (m, 1H), 4.50 (m, 1H), 4.13 (d,0.5H, J=5.2 Hz), 3.97 (d, 0.5H, J=5.5 Hz), 3.46 (m, 2H), 3.02 (m, 3H),2.43 (s, 1/2×3H), 2.42 (s, 1/2×3H), 2.07 (m, 1H), 1.45 (m, 1H). ¹³C NMR(DMSO-d₆): δ=172.9, 172.8, 170.7, 170.7, 144.1, 143.9, 141.4, 141.3,138.0, 137.7, 134.9, 134.6, 130.2, 130.1, 129.8, 129.6, 128.7, 128.6,128.5, 127.8. 127.7, 127.2, 127.1, 126.9, 126.8, 126.7, 79.6, 67.9,53.6, 53.5, 49.7, 49.5, 49.5, 49.1, 37.2, 37.1, 32.5, 32.3, 21.4. MassSpectroscopy: FAB m/e 493 (M+H).

Example 25 Synthesis ofN-(3,4-Dimethoxybenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as in Example 21 (59) except that3,4-dimethoxyphenylsulfonyl chloride was used in place of the3,5-dichlorophenylsulfonyl chloride. NMR analysis indicated thatepimerization had occurred to give a ca. 65:35 mixture of diastereomers.

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.46 (m, 1H), 7.39-7.20 (6H),6.97 (dd, 1H, J=4.5, 8.4 Hz), 4.85 (m, 1H), 4.10 (m, 1H), 3.96 (s,0.35×3H), 3.95 (s, 0.65×3H), 3.93 (s, 0.35×3H), 3.92 (s, 0.65×3H),3.44-3.22 (2H), 3.11 (m, 2H), 2.01 (m, 1H), 1.60-1.33 (3H). ¹³C NMR(CDCl₃): δ=175.1, 174.7, 172.5, 172.3, 153.7, 149.8, 136.6, 135.9,130.2, 129.9, 129.4, 129.1, 128.0, 128.0, 127.9, 127.7, 122.4, 111.4,110.7, 63.2, 62.8, 57.0, 56.8, 53.8, 53.6, 50.3, 50.2, 38.0, 31.1, 30.5,24.8, 24.7. Mass Spectroscopy: FAB m/e 463 (M+H).

Example 26 Synthesis ofN-(Benzene-4-sulfonyl)-D,L-(cis-3-phenyl)prolyl-L-phenylalanine

N-Acetyl-cis-3-phenylproline ethyl ester (614 mg, 2.35 mmol) (preparedvia the method of Chung, et al., J. Org. Chem. 55: 270-275 (1990)) wasdissolved in HOAc (4 mL) and 6N HCl (12 mL) and heated at reflux for 18hr. The mixture was cooled to room temperature and the volatiles wereevaporated in vacuo to give a white solid. The solid was dissolved in 1NNaOH (15 mL) and dioxane was added (10 mL), followed by4-toluene-sulfonyl chloride (458 mg, 2.40 mmol). The mixture was stirredat room temp for 18 hr before addition of 6N HCl to bring the pH<4. Themixture was extracted with Et₂O (3×40 mL) and the extracts were dried(MgSO₄), filtered, and evaporated in vacuo to giveN-(toluene-4-sulfonyl)-cis-3-phenylproline as a white solid (765 mg,94%).

N-(Toluene-4-sulfonyl)-cis-3-phenylproline was coupled to phenylalanineethyl ester using the procedure described in Method 3. The titlecompound was prepared via hydrolysis of the ethyl ester using NaOH inethanol.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.57 (bs, 1H), 7.77 (m, 2H),7.31-6.70 (12H), 4.65 (m, 0.5H), 4.47 (m, 0.5H), 4.41 (d, 0.5H, J=8.9Hz), 4.34 (d, 0.5H, J=9.0 Hz), 3.73, (m, 1H), 3.28-2.76 (4H), 2.44 (s,3H), 2.40 (m, 0.5H), 2.17 (m, 0.5H), 1.8 (m, 1H). ¹³C NMR (CDCl₃):δ=174.5, 173.9, 169.5, 168.8, 144.3, 136.1, 135.8, 135.6, 135.3, 133.4,133.1, 129.9, 129.6, 129.4, 128.6, 128.6, 128.3, 127.8, 127.7, 127.5,127.1, 127.0, 65.9, 65.8, 53.6, 52.5, 48.5, 48.3, 48.2, 37.5, 37.3,39.0, 28.6, 21.6. Mass Spectroscopy: FAB m/e 493 (M+H).

Example 27 Synthesis ofN-(4-Nitrobenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as in Example 2.1 (59) except4-nitrophenylsulfonyl chloride was used in place of3,5-dichlorophenylsulfonyl chloride, mp=70-80° C.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.38 (d, 2H J=18.9 Hz), 8.02(d, 2H, J=8.9 Hz), 7.32-7.20 (6H), 4.90 (m, 1H), 4.25 (dd, 1H, J=2.5,8.4 Hz), 3.42 (m, 2H), 3.16 (m, 2H), 1.98 (m, 3H), 1.62 (m, 2H), 1.54(m, 1H). ¹³C NMR (CDCl₃): δ=175.0, 171.8, 151.1, 142.5, 136.4, 129.9,129.7, 127.8, 125.2, 62.9, 53.7, 50.3, 37.9, 30.9, 24.7. MassSpectroscopy: FAB m/e 448 (M+H).

Example 28 Synthesis ofN-(4-Acetamidobenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as in Example 21 (59) except4-acetamido-phenylsulfonyl chloride is used in place of3,5-dichlorophenylsulfonyl chloride.

NMR data was as follows: ¹H NMR (DMSO-d₆): δ=12.85 (ds, 1H), 10.4 (bs,1H), 8.07 (d, 1H, J=8.2 Hz), 7.76 (ab q, 4H), 7.25 (m, 5H), 4.49 (dd,2H, J=3.2, 8.2 Hz), 4.06 (m, (H), 3.36 (m, 1H), 3.08 (3H), 2.09 (s, 3H),1.58-1.48 (4H). ¹³C NMR (DMSO-d₆): δ=173.0, 171.2, 169.5, 143.9, 137.8,130.4, 129.7, 129.1, 128.5, 126.8, 119.0, 61.7, 53.5, 49.4, 37.0, 30.7,24.6, 24.1. Mass Spectroscopy: FAB m/e 460 (M+H).

Example 29 Synthesis ofN-(4-Cyanobenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared as in Example 21 (59) except4-cyanophenyl-sulfonyl chloride is used in place of3,5-dichlorophenylsulfonyl chloride.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.40 (bs, 1H), 7.95 (d, 2H,J=8.3 Hz), 8.07 (d, 2H, J=8.3 Hz), 7.36-7.20 (6H), 4.93, (dt, 1H, J=5.4,8.0 Hz), 4.22 (m, 1H), 3.38 (m, 2H), 3.13 (m, 2H), 1.92 (m, 1H), 1.56(m, 2H), 1.40 (m, 1H). ¹³C NMR (CDCl₃): δ=175.0, 171.9, 140.8, 136.5,133.8, 129.9, 129.2, 129.0, 127.8, 117.8, 117.7, 62.8, 53.7, 50.3, 37.9,30.9, 24.6. Mass Spectroscopy: FAB m/e 428 (M+H).

Example 30 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-tryptophan

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled to L-tryptophanmethyl ester hydrochloride using the procedure described in Method 3.The title compound was prepared via hydrolysis of the methyl ester usingLiOH in THF/water.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.45 (d, 1H, J=2.0 Hz), 7.64(d, 2H, J=8.2 Hz), 7.60 (d, 1H, J=7.6 Hz), 7.45 (d, 1H, J=7.4 Hz), 7.33(d, 1H, J=8.0 Hz), 7.27 (d, 2H, J=8.0 Hz), 7.19-7.06 (3H), 4.88 (m, 1H),4.07 (dd, 1H, J=2.9, 8.6 Hz), 3.48 (dd, 1H, J=5.4, 14.8 Hz), 3.36 (dd,1H, J=7.1, 14.8 Hz), 3.03 (m, 2H), 2.40 (s, 3H), 1.88 (m, 1H), 1.47 (m,1H), 1.32 (m, 1H), 1.22 (m, 1H). ¹³C NMR (CDCl₃): δ=174.8, 172.7, 145.0,136.7, 133.3, 130.6, 128.4, 128.3, 124.3, 122.6, 118.9, 112.0, 110.0,62.8, 54.0, 50.1, 30.5, 27.5, 24.6, 22.1. Mass Spectroscopy: FAB m/e 456(M+H).

Example 31 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-β-(1-naphthyl)-L-alanine

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled toβ-(1-naphthyl)alanine methyl ester hydrochloride using the proceduredescribed in Method 3. The title compound was prepared via hydrolysis ofthe methyl ester using LiOH in THF/water.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.16 (d, 1H, J=8.2 Hz), 8.05(bs, 1H), 7.85 (d, 1H, J=6.9 Hz), 7.77-7.27 (10H), 4.99 (m, 1H), 4.05(m, 1H), 3.90 (dd, 1H, J=5.0, 14.3 Hz), 3.51 (dd, 1H, J=9.6, 14.3 Hz),3.11 (m, 1H), 3.02 (m, 1H), 2.41 (s, 3H), 1.74 (m, 1H), 1.34 (m, 2H),1.00 (m, 1H). ¹³C NMR (CDCl₃): δ=174.9, 172.5, 145.0, 134.4, 133.4,133.1, 132.6, 130.6, 129.4, 128.5, 128.4, 127.2, 126.4, 126.0, 14.0,62.7, 53.9, 50.1, 34.9, 30.3, 24.5, 22.1. Mass Spectroscopy: FAB m/e 467(M+H).

Example 32 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-β-(2-naphthyl)-L-alanine

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled toβ-(2-naphthyl)-L-alanine methyl ester hydrochloride using the proceduredescribed in Method 3. The title compound was prepared via hydrolysis ofthe methyl ester using LiOH in THF/water.

NMR data was as follows ¹H NMR (CDCl₃): δ=8.20 (bs, 1H), 7.80-7.67 (6H),7.55 (d, 1H, J=7.9 Hz), 7.46-7.27 (5H), 4.99 (m, 1H), 4.12 (m, 1H), 3.55(m, 1H), 3.27 (dd, 1H, J=8.2, 14.1 Hz), 3.17 (m, 1H), 3.01 (m, 1H), 2.39(s, 3H), 1.83 (m, 1H), 1.40-1.26 (3H). ¹³C NMR (CDCl₃): δ=174.8, 172.4,145.0, 134.3, 133.9, 133.4, 133.0, 130.6, 128.9, 128.8, 128.4, 128.2,128.1, 127.9, 126.7, 126.3, 62.7, 53.9, 50.1, 38.2, 30.4, 24.5, 22.1.Mass Spectroscopy: FAB m/e 467 (M+H).

Example 33 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-β-(2-thienyl)-L-alanine

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled toβ-(2-thienyl)-L-alanine methyl ester hydrochloride using the proceduredescribed in Method 3. The title compound was prepared via hydrolysis ofthe methyl ester using LiOH in THF/water.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.42 (bs, 1H), 7.13 (d, 2H,J=8.3 Hz), 7.57 (d, 1H, J=7.4 Hz), 7.34 (d, 2H, J=7.9 Hz), 7.16 (dd, 1H,J=1.2, 5.1 Hz), 6.95-6.88 (m, 2H), 4.87 (m, 1H), 4.18 (m, 1H) 3.56-3.41(3H), 3.17 (m, 1H), 2.43 (s, 3H), 2.08 (m, 1H), 1.60 (m, 3H). ¹³C NMR(CDCl₃): δ=174.2, 172.6, 145.1, 137.9, 133.4, 130.6, 128.5, 127.7,127.6, 125.4, 62.8, 54.0, 50.4, 32.11, 30.7, 24.9, 22.2 MassSpectroscopy: FAB m/e 423 (M+H).

Example 34 Synthesis of N-(Isopropanesulfonyl)-L-prolyl-L-phenylalanine

Substitution of (CH₃)₂CHSO₂Cl for CH₃SO₂Cl, and following the proceduresdescribed in Example 1 (9), gave the title compound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.10 (d, J=7.9,1H), 7.28-7.16 (m, 5H), 4.48-4.41 (m, 1H), 4.27-4.24 (m, 1H), 3.47-3.23(m, 3H), 3.11-3.00 (m, 2H), 2.89 (dd, J=13.9, J=9.4, 1H), 2.11-2.01 (m,1H), 1.82-1.71 (m, 3H), 1.11 (d, J=6.8, 6H). ¹³C NMR (DMSO-d₆, 75 MHz):δ=172.6, 171.1, 137.4, 129.2, 128.1, 126.4, 61.2, 53.0, 48.6, 43.6,36.5, 30.6, 24.0, 20.0, 19.8. Mass Spectroscopy: (+FAB, 3-nitrobenzylalcohol) 369 (MH+).

Example 35 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-β-(3-pyridyl)-L-alanine

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled toL-β-(3-pyridyl)alanine methyl ester dihydrochloride using the proceduredescribed in Method 3 to giveN-(toluene-4-sulfonyl)prolyl-L-β-(3-pyridyl)alanine. The title compoundwas prepared via hydrolysis of the methyl ester using 0.5 N aqueous NaOHin THF/water.

NMR data was as follows: ¹H NMR (DMSO-d₆): δ=8.33 (dd, 1H, J=1.4, 4.7Hz), 8.27 (d, 1H, J=1.9 Hz), 7.75 (d, 2H, J=8.2 Hz), 7.71 (m, 1H), 7.52(m, 1H), 7.42 (d, 2H, J=8.5 Hz), 7.19 (dd, 1H, J=4.7, 7.7 Hz), 4.00 (m,2H), 3.20-3.00 (4H), 2.40 (s, 3H), 1.72 (m, 1H), 1.40 (3H). ¹³C NMR(DMSO-d): δ=172.7, 170.0, 150.9, 147.4, 144.1, 137.4, 134.6, 133.8,130.3, 128.1, 123.1, 62.4, 55.1, 49.3, 34.4, 30.5, 24.00, 21.4. MassSpectroscopy: FAB m/e 440 (M+Na).

Example 36 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-phenylthio)prolyl-L-phenylalanine

Trans-4-hydroxy-L-proline was treated with EtOH and HCl gas, and themixture was evaporated to give trans-4-hydroxy-L-proline ethyl esterhydrochloride. This product was treated with TsCl in pyridine, to giveafter aqueous workupN-(toluene-4-sulfonyl)-trans-4-(O-(toluene-4-sulfonyl))-L-proline ethylester. This product was treated with PhSH and DBU in DMF to give afteraqueous workup and flash chromatography on silica gel,N-(Toluene-4-sulfonyl)-cis-4-(phenylthio)-L-proline ethyl ester. Thisproduct was treated with NaOH in CH₃OH and H₂O, to give afteracidification, extraction, drying with MgSO₄, filtration andevaporation, N-(toluene-4-sulfonyl)-cis-4-(phenylthio)-L-proline. Thisproduct was treated with HCl.Phe-OMe, BOP, and NMM in DMF, to give,after aqueous workup and flash chromatography,N-(toluene-4-sulfonyl)-cis-4-(phenylthio)-Pro-Phe-OMe. This product wastreated with NaOH in CH₃OH and H₂O, to give, after acidification,extraction, drying with MgSO⁴, filtration and evaporation, the titlecompound as a clear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.19 (d, J=8.0,1H), 7.71 (d, J=8.3, 2H), 7.41 (d, J=8.2, 2H), 7.35-7.14 (m, 10H),4.534.46 (m, 1H), 4.24 (t, J=7.8, 1H), 3.73 (dd, J=11.6, J=6.7, 1H),3.36 (bs, 1H), 3.17-2.86 (m, 4H), 2.39 (s, 3H), 2.34-2.25 (m, 1H),1.57-1.47 (m, 1H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.4, 170.0, 144.0,137.3, 134.1, 133.9, 130.0, 129.8, 129.4, 129.3, 128.2, 127.5, 126.9,126.5, 61.0, 54.9, 53.2, 42.3, 36.7, 36.4, 21.1. Mass Spectroscopy:(+FAB, 3-nitrobenzyl alcohol) 525 (MH+).

Example 37 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-benzylthio)prolyl-L-phenylalanine

Substitution of benzylthiol for phenylthiol, and following the methodsfor preparation of Example 36 (131), gave the title compound as a clearoil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.18 (d, J=8.1,1H), 7.65 (d, J=8.3, 2H), 7.38 (d, J=8.2, 2H), 7.29-7.17 (m, 10H),4.50-4.43 (m, 1H), 4.05 (t, J=7.9, 1H), 3.74-3.56 (m, 3H), 3.35 (bs,1H), 3.09-2.94 (m, 4H), 2.40 (s, 3H), 2.20-2.10 (m, 1H), 1.40-1.28 (m,1H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.4, 170.2, 143.8, 138.5, 137.3,134.0, 130.0, 129.4, 128.7, 128.4, 128.2, 127.3, 126.9, 126.5, 60.9,55.5, 53.1, 39.3, 36.7, 36.6, 34.9, 21.1. Mass Spectroscopy: (+FAB,3-nitrobenzyl alcohol) 539 (MH+).

Example 38 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-histidine

N-(Toluene-4-sulfonyl)prolyl-L-(N-benzyl)histidine (237 mg, 0.464 mmol)was dissolved in MeOH (20 mL) and 10% Pd/C (50 mg) was added. Themixture was hydrogenated at 50 psi H₂ for 36 hr. The mixture wasfiltered to remove the catalyst and the filtrate was evaporated invacuo. The residue was purified by preparative TLC (90:10:1CH₂Cl₂/MeOH/NH₄OH) to give N-(toluene-4-sulfonyl)-L-prolyl-L-histidinemethyl ester (155 mg, 79%). The title compound was prepared viahydrolysis of the methyl ester using 0.5 N NaOH in THF/water (128 mg,81%).

NMR data was as follows: ¹H NMR (DMSO_(d) ₆): δ=7.77 (d, 3H, J=6.5 Hz),7.42 (m, 3H), 4.03 (m, 1H), 3.99 (m, 1H), 3.22 (m, 1H), 3.05 (m 2H),2.95 (m, 1H), 2.41 (s, 3H), 1.80 (m, 1H), 1.45 (3H). ¹³C NMR (DMSO-d₆):δ=174.0, 169.8, 144.1, 133.8, 10.3, 128.0, 62.4, 49.4, 40.0, 30.6, 24.0,21.4. Mass Spectroscopy: FAB m/e 429 (M+Na).

Example 39 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-amino)prolyl-L-phenylalanine

Trans-4-hydroxy-L-proline was treated with EtOH and HCl gas, and themixture was evaporated to give trans-4-hydroxy-L-proline ethyl esterhydrochloride. This product was treated with TsCl in pyridine to give,after aqueous workup, N-(toluene-4-sulfonyl)-trans-4-(TsO)-L-prolineethyl ester. This product was treated with NaN₃ in DMF and H₂O to give,after aqueous workup and flash chromatography,N-(toluene-4-sulfonyl)-cis-4-azido-L-proline ethyl ester. This productwas treated with NaOH in CH₃OH and H₂O to give, after acidification,extraction, drying with MgSO₄, filtration and evaporation,N-(toluene-4-sulfonyl)-cis-4-azido-L-proline. This product was treatedwith 10% Pd on C in THF, AcOH and H₂O, and the mixture was shaken under50 psi H₂. The mixture was filtered through Celite and evaporated togive N-(toluene-4-sulfonyl)-cis-4-amino-L-proline. This product wastreated with Boc₂O and Et₃N in t-BuOH and H₂O to give, afterevaporation, acidification, extraction, drying with MgSO₄, filtration,and evaporation,N-(toluene-4-sulfonyl)-cis-4-(t-butoxycarbonylamino)-L-proline. Thisproduct was treated with HCl.Phe-O-t-Bu, BOP, arid NMM in DMF to give,after aqueous workup and flash chromatography,N-(toluene-4-sulfonyl)-cis-4-(t-butoxycarbonylamino)-Pro-Phe-O-t-Bu.This product was treated with TFA to give after evaporation thetrifluoroacetate salt of the title compound as a clear oil.

NMR data was as follows: ¹H NMR (CD₃OD, 300 MHz): δ=7.47 (d, J=8.3, 2H),7.22-7.00 (m, 7H), 4.47 (dd, J=7.9, J=5.3, 1H), 4.18 (dd, J.=9.8, J=1.7,1H), 3.64 (t, J=5.2, 1H), 3.43 (d, J=11.2, 1H), 3.22 (dd, J=11.3, J=5.0,1H), 3.01 (dd, J=14.1, J=5.3, 1H), 2.88 (dd, J=14.0, J=7.9, 1H), 2.23(s, 3H), 2.21-2.08 (m, 1H), 1.87-1.80 (m, 1H). ¹³C NMR (CD₃OD, 75 MHz):δ=174.7, 173.9, 146.1, 138.0, 134.7, 131.1, 130.5, 129.6, 128.7, 128.0,60.7, 55.6, 53.9, 51.5, 38.2, 35.3, 21.5. Mass Spectroscopy: (+FAB,3-nitrobenzyl alcohol) 432 (MH+).

Example 40 Synthesis ofN-(Toluene-4-sulfonyl)-L-(trans-4-amino)prolyl-L-phenylanine

Substitution of cis-4-hydroxy-L-proline for trans-4-hydroxy-L-proline,and following the methods for preparation of Example 39 (136), gave thetrifluoroacetate salt of the title compound as a clear oil.

NMR data was as follows: ¹H NMR (CD₃OD, 300 MHz): δ=7.39 (d, J=8.4, 2H),7.13-7.01 (m, 7H), 4.404.33 (m, 1H), 4.28 (dd, J=9.0, J=2.2, 1H),3.59-3.50 (m, 2H), 3.07-2.97 (m, 2H), 2.81 (dd, J=14.0, J=8.9, 1H), 2.21(s, 3H), 1.24-2.06 (m, 1H), 1.80-1.68 (m, 1H). ¹³C NMR (CD₃OD, 75 MHz):δ=174.1, 172.6, 145.9, 138.3, 135.0, 131.0, 130.5, 129.6, 128.9, 128.0,61.1, 55.2, 52.0, 49.7, 38.2, 35.4, 21.5. Mass Spectroscopy: (+FAB,3-nitrobenzyl alcohol) 432 (MH+).

Example 41 Synthesis of N-(Toluenesulfonyl)-L-prolyl-L-phenylalanineBenzyl Ester

N-(Toluene-4-sulfonyl)-L-proline was coupled to L-phenylalanine benzylester toluenesulfonic acid salt using the procedure described in Method3 to give the title compound as an oil.

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.68 (d, 2H, J=8.2 Hz),7.39-7.17 (10H), 7.05 (m, 1H), 5.22 (d, 2H, J=12.2 Hz), 5.14 (d, 1H,J=12.2 Hz), 4.88 (m, 1H), 4.08 (m, 1H), 3.28 (m, 3H), 3.08 (m, 2H), 2.39(s, 3H), 2.00 (m, 1H), 1.46 (m, 3H). ¹³C NMR (CDCl₃): δ=171.4,171.3,-144.9, 136.5, 135.8, 133.6, 130.6, 129.9, 129.1, 129.1, 129.0,129.0, 128.4, 127.6, 67.8, 62.8, 54.0, 50.2, 38.4, 30.4, 24.8, 22.2.Mass Spectroscopy: FAB m/e 507 (M+H).

Example 42 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-Methoxyamide

N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanine was coupled toO-methylhydroxylamine hydrochloride using the mixed anhydride procedure(as described in Greenstein, J. P. and Milton Wenitz, “Chemistry of theAmino Acids,” volume 2, pp. 978-979, Robert E. Krieger Publishing Co.,Malabar, Fla. (1961)) to give the title compound as an oil.

NMR data was as follows: ¹H NMR (CDCl₃): δ=9.66 (s, 1H), 7.70 (d, 2H,J=8.2 Hz), 7.36 (d, 2H, J=8.0 Hz), 7.33-7.21 (5H), 6.90 (d, 1H, J=9.5Hz), 4.88 (m, 1H), 3.81 (m, 1H), 3.75 (s, 3H), 3.46 (m, 2H), 3.07 (m,2H), 2.45 (s, 3H), 1.60 (m, 2H), 1.47 (m, 2H). ¹³C NMR (CDCl₃): δ=171.7,168.1, 145.6, 137.0, 131.8, 130.7, 129.7, 129.3, 128.5, 127.7, 64.9,62.7, 52.6, 50.6, 37.6, 31.4, 24.7, 22.2. Mass Spectroscopy: FAB m/e 446(M+H).

Example 43 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-Benzyloxyamide

N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanine was coupled toO-benzylhydroxylamine using the procedure described in Method 3 to givethe title compound as a white solid, mp=127-130° C.

NMR data was as follows: ¹H NMR (CDCl₃): δ=9.54 (s, 1H), 7.61 (d, 2H,J=8.2 Hz), 7.43-7.19 (12H), 6.86 (d, 1H, J=9.3 Hz), 4.91 (m, 2H), 4.82(m, 1H), 3.72 (m, 1H), 3.42 (m, 2H), 3.07 (m, 2H), 2.45 (s, 3H), 1.64(m, 2H), 1.44 (m, 1H), 1.26 (m, 1H). ¹³C NMR (CDCl₃): δ=171.6, 168.2,145.5, 136.9, 135.7, 132.0, 130.7, 129.9, 129.7, 129.0, 128.9, 128.5,127.7, 78.8, 62.7, 52.7, 50.6, 37.6, 31.2, 24.7, 22.2. MassSpectroscopy: FAB m/e 522 (M+H).

Example 44 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-(Toluene-4-sulfonyl)amide

N-(Toluene-4-sulfonyl)-L-proline was reacted with L-phenylalanine amideusing the procedure described in Method 12 to yieldN-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine amide. To a solution ofNaH (60% mineral oil—prewashed with THF) in THF at 0° C. was addedN-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine amide and the reactionwas stirred at 0° C. for 45 minutes. Toluene-4-sulfonyl chloride wasadded and the reaction was stirred for 16 hours at room temperature. Thereaction mixture was extracted with EtOAc (3×50 mL) and 0.2 N HCl (50mL), and the combined organic layers were washed successively with sat.NaHCO (50 mL), and sat. NaCl (2×50 mL), dried over MgSO₄, filtered, andconcentrated to yield the title compound as an oil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.97 (d, 2H, 8.52Hz); 7.72 (d, 2H, 8.52 Hz); 7.37-7.04 (m, 10 H); 4.80 (m, 1H); 3.86 (m,1H); 3.42 (m, 1H); 3.29 (m, 1H); 3.01 (m, 2H); 2.45 (bs, 6H); 1.65 (m2H); 1.45 & 1.31 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): δ=172.46, 169.93,145.47, 136.43, 132.06, 130.70, 130.07, 129.58, 129.32, 129.17, 128.61,127.74, 62.68, 54.52, 50.57, 36.91, 31.10, 24.69, 22.30, 22.23. MassSpectroscopy: (+FAB) 570 (M+H).

Preparative Example A Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-D,L-phenyalanyl-β-alanine Ethyl Ester

To N-(toluene-4-sulfonyl)-L-prolyl-L-phenylalanine (˜2 eq.) in DMF wasadded BOP (˜2.1 eq.) and NMM (˜4 eq.), and the reaction was stirred atroom temperature for about 45 minutes. β-alanine ethyl ester (˜2 eq.)was added and the reaction stirred for about 16 hours at roomtemperature. The reaction mixture was extracted with water and diethylether. The combined organic solution was then washed with 0.2 N HCl,sat. NaHCO₃ and sat. NaCl solutions. The organic layer was dried overanhydrous magnesium sulfate, filtered and concentrated. The residue waspurified by silica gel chromatography (50% EtOAc/Hexane, Rf=0.18) toyield the title compound as a colorless oil, which was determined to bea mixture of diastereomers, by NMR.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.68 (m, 2H); 7.25(m, 9H); 4.07 (m, 3H); 3.45 (m, 4H); 3.06 (m, 3H); 2.50 & 2.34 (m, 3H),2.41 (s, 3H), 1.62 (m, 4H); 1.20 (t, 3H, J=5.55 Hz). ¹³C NMR (CDCl₃, 300MHz): δ=177.62, 172.72, 171.98, 171.73, 171.25, 170.80, 145.39, 145.02,137.52, 137.20, 133.28, 132.09, 133.28, 132.09, 130.66, 130.57, 130.02,129.59, 129.18, 128.49, 128.44, 127.49, 127.46, 63.06, 62.75, 61.18,61.12, 55.49, 54.17, 50.62, 50.36, 38.27, 37.92, 35.98, 35.32, 34.47,34.30, 31.27, 30.95, 24.92, 24.62, 22.18, 14.72. Mass Spectroscopy:(+FAB) 516 (M+H).

Example 45 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-D,L-phenyalanyl-β-alanine

The title compound was prepared from Preparative Example A (161) usingthe procedure described for Example 19 (57), yielding a translucentsolid, which was determined to be a mixture of diastereomers by NMR,mp=91-95° C.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.70 & 7.27 (m, 9H);4.83 (m, 0.5H); 4.05 (m, 0.5H); 3.47 (m, 4H); 3.06 (m, 3H); 2.55 (m,2H); 2.43 (s, 3H); 1.62 (m, 4H). ¹C NMR (CDCl₃, 300 MHz): δ=175.85,175.68, 172.45, 172.23, 171.67, 171.43, 145.32, 144.96, 137.37, 136.91,133.42, 132.37, 130.67, 130.56, 130.52, 130.03, 129.65, 129.15, 129.12,128.51, 128.48, 127.52, 63.01, 62.72, 55.25, 54.34, 50.52, 50.43, 50.39,38.81, 38.07, 35.82, 35.79, 35.25, 35.23, 34.17, 34.10, 34.08, 31.23,24.91, 24.61, 22.18. Mass Spectroscopy: (+FAB) 488 (M+H).

Example 46 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-Hydroxyamide

N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanine N-benzyloxyamide (seeExample 43 (159)) was dissolved in MeOH (10 mL) and 5% Pd/BaSO₄ (52 mg)was added. The mixture was hydrogenated at 50 psi H₂ for 9 h. Themixture was filtered through a pad of diatomaceous earth and evaporatedin vacuo to give a residue which was purified by silica gelchromatography (92:8 CH₂Cl₂/MeOH) to give the title compound as an oil.

NMR data was as follows: ¹H NMR (CDCl₃): δ=9.85 (br s, 1H), 7.73 (d, 2H,J=7.2 Hz), 7.36 (d, 2H, J=7.0 Hz), 7.28-7.20 (5H), 7.07 (m, 1H), 4.96(m, 1H), 3.87 (m, 1H), 3.47 (m, 2H), 3.07 (m, 2H), 2.45 (s, 3H), 1.90(br s, 1H), 1.63 (m, 2H), 1.46 (m, 1H), 1.36 (m, 1H). ¹³C NMR (CDCl₃):δ=171.8, 168.4, 145.5, 136.9, 132.0, 130.7, 129.7, 129.2, 128.6, 127.6,62.7, 52.2, 50.6, 38.0, 31.2, 24.6, 22.2. Mass Spectroscopy: FAB m/e 432(M+H).

Example 47 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineIsopropyl Ester

N-(Toluene-4-sulfonyl)-L-proline hydrate was coupled to phenylalanineisopropyl ester trifluoroacetate using the procedure described in Method3 to give the title compound as an oil.

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.68 (d, 2H, J=8.2 Hz),7.33-7.12 (8H), 5.05 (p, 1H, J=6.3 Hz), 4.77 (m, 1H), 4.06 (m, 1H), 3.33(m, 1H), 3.22 (dd, 1H, J=5.8, 14.0 Hz), 3.06 (m, 2H), 2.40 (s, 3H), 1.99(m, 1H), 1.46 (3H), 1.23 (d, 6H, J=6.3 Hz). ¹³C NMR (CDCl₃): δ=171.3,170.9, 144.9, 136.7, 133.5, 130.5, 129.9, 129.0, 128.4, 127.5, 70.0,62.9, 54.0, 50.2, 38.5, 30.4, 24.7, 22.3, 22.2, 22.1. Mass Spectroscopy:FAB m/e 459 (M+H).

Example 48 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-hydroxy)-prolyl-L-phenylalanine

Substitution of cis-4-hydroxy-L-proline for D-Pro-OH, and following themethods described in Example 6 (17), gave the title compound as a clearoil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.14 (d, J=7.9,1H), 7.70 (d, J=8.2, 2H), 7.41 (d, J=8.2, 2H), 7.28-7.15 (m, 5H), 5.30(bs, 1H), 4.52-2.45 (m, 1H), 4.15 (dd, J=9.0, J=4.1, 1H), 3.85 (bs, 1H),3.31 (bs, 1H), 3.24 (dd, J=10.6, J=4.9, 1H), 3.17 (dd, J=10.9, J=3.4,1H), 3.01-2.94 (m, 2H), 2.40 (s, 3H), 1.92-1.82 (m, 1H), 1.79-1.69 (m,1H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.2, 171.2, 143.8, 136.9, 133.4,129.9, 129.5, 128.1, 127.5, 126.5, 68.6, 60.3, 56.4, 53.4, 38.0, 37.0,21.0. Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 433 (MH+).

Example 49 Synthesis ofN-(Toluene-4-sulfonyl)-L-(trans-4-fluoro)-prolyl-L-phenylalanine BenzylEster

Substitution of cis-4-hydroxy-L-proline for trans-4-hydroxy-L-proline,and following the methods for preparation of Example 50 (177), gave thetitle compound as a clear oil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.70 (d, J=8.3, 2H),7.41-7.20 (m, 10H), 7.10-7.07 (m, 2H), 5.24 (dd, J=12.1, 1H), 5.16 (dd,J=12.2, 1H), 4.934.87 (m, 1H), 4.88 (bd, J=52.4, 1H), 4.154.09 (m, 1H),3.80 (ddd, J=1.6, J=20.7, J=12.5, 1H), 3.37 (ddd, J=3.1, J=13.9, J=36.8,1H), 3.28 (dd, J=13.9, J=5.8, 1H), 3.06 (dd, J=13.9, J=7.2, 1H), 2.43(s, 3H), 2.32-2.18 (m, 1H), 2.11-1.91 (m, 1H). ¹³C NMR (CDCl₃, 75 MHz):δ=170.7, 170.0, 144.4, 135.8, 135.1, 129.7, 129.4, 128.58, 128.56,128.4, 128.1, 127.0, 91.2 (d, J=181.0 Hz), 67.3, 60.8, 55.6 (d, J=23.0Hz), 53.2, 37.9, 37.0 (d, J=22.0 Hz), 21.6. Mass Spectroscopy: (+FAB,3-nitrobenzyl alcohol) 525 (MH+).

Example 50 Synthesis ofN-(Toluene-4-sulfonyl)-L-(cis-4-fluoro)-prolyl-L-phenylalanine BenzylEster

Trans-4-hydroxy-L-proline was treated with EtOH and HCl gas, and themixture was evaporated to give trans-4-hydroxy-L-proline ethyl esterhydrochloride. This product was treated with TsCl and Et₃N in CH₂Cl₂, togive after aqueous workupN-(toluene-4-sulfonyl)-trans-4-hydroxy-L-proline ethyl ester. Thisproduct was treated with morpholinosulfur trifluoride in CH₂Cl₂, to giveafter aqueous workup N-(toluene-4-sulfonyl)-cis-4-fluoro-L-proline ethylester. This product was treated with NaOH in CH₃OH and H₂O to give,after acidification, extraction, drying with MgSO⁴, filtration andevaporation, N-(toluene-4-sulfonyl)-cis-4-fluoro-L-proline. This productwas treated with HCl.Phe-OBn, BOP, and NMM in DMF, to give after aqueousworkup and flash chromatography the title compound as a clear oil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.69 (d, J=8.3, 2H),7.41 (d, J=7.8, 1H), 7.38-7.27 (m, 7H), 7.21-7.17 (m, 3H), 7.07-7.04 (m,2H), 5.19 (d, J=12.1, 1H), 5.12 (d, J=12.1, 1H), 5.05 (dt, Jd=52.7,Jt=3.4, 1H), 4.89 (dt, Jd=7.9, Jt=6.0, 1H), 4.27 (d, J=9.9, 1H), 3.68(ddd, J=1.6, J=12.6, J=21.1, 1H), 3.37 (ddd, J=3.7, J=12.4, J=35.7, 1H)3.13 (dd, J=5.9, J=13.8, 1H), 3.08 (dd, J=6.1, J=13.8, 1H), 2.62 (t,J=15.9, 1H), 2.43 (s, 3H), 1.82-1.59 (m, 1H). ¹³C NMR (CDCl₃, 75 MHz):δ=170.5, 169.9, 144.8, 135.5, 135.1, 132.4, 130.1, 129.41, 129.39,128.6, 128.5, 128.4, 127.9, 126.9, 91.7 (d, J=179.4 Hz), 67.1, 55.6 (d,J=23.8 Hz), 36.3 (d, J=21.5 Hz), 21.6. Mass Spectroscopy: (+FAB,3-nitrobenzyl alcohol) 525 (MH+).

Example 51

Synthesis of N-(Toluene-4-sulfonyl)-L-thiaprolyl-L-phenylalanine BenzylEster

The title compound was prepared following the procedure outlined for thepreparation of Example 3 (11).

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=2.45 (s, 3H),3.05-3.35 (m, 2H), 4.03 (d, 1H, J=10.3 Hz), 4.55 (d, 1H, J=10.3 Hz),4.60 (m, 1H), 4.86 (m, 1H), 5.20 (dd, 2H, J=12.1 and 13.6 Hz), 7.03 (m,1H), 7.14-7.40 (m, 12H), 7.70 (d, 2H, J=8.2 Hz). ¹³C NMR (CDCl₃, 75MHz): δ=22.3, 33.5, 38.1, 52.0, 54.2, 65.8, 68.0, 127.8, 128.5, 129.1,129.2, 129.9, 130.7, 134.0, 135.6, 136.0, 145.6, 168.7, 171.1. MassSpectroscopy: (FAB+) 525 (M+H).

Example 52 Synthesis ofN-(Toluene-4-sulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanine BenzylEster

The title compound was prepared following the procedure described forthe preparation of Example 3 (11).

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=1.10 (s, 6H), 2.44(s, 3H), 3.08-3.14 (m, 2H), 3.85 (s, 1H), 4.39 (d, 1H, J=9.7 Hz), 4.52(d, 1H, J=9.7 Hz), 4.94 (m, 1H), 5.17 (m, 2H), 7.01 (d, 1H, J=2.5 Hz),7.10-7.40 (m, 12H), 7.74 (d, 2H, J=8.3 Hz). ¹³C NMR (CDCl₃, 75 MHz):δ=22.3, 24.5, 29.8, 38.9, 51.2, 53.9, 55.2, 67.9, 74.1, 127.6, 128.7,129.0, 129.1, 129.2, 129.3, 130.0, 130.5, 133.3, 135.7, 136.4, 145.3,169.0, 171.3. Mass Spectroscopy: (FAB+) 553 (M+H).

Example 53 Synthesis ofN-(2-Methoxycarbonylbenzenesulfonyl)-L-prolyl-L-phenylalanine BenzylEster

To L-prolyl-L-phenylalanine benzyl ester (4.64 mmoles) in THF (15 mL)was added triethylamine (4.70 mmoles) and the reaction proceeded for 30minutes at room temperature. The reaction mixture was chilled to 0° C.and methyl 2-(chlorosulfonyl) benzoate (4.64 mmoles) was added and thereaction proceeded for 4 hours at room temperature. The reaction wasextracted with EtOAc (3×50 mL) and water (50 mL), and the combinedorganic layers were successively washed with sat. NaHCO₃ (50 mL) and satNaCl (2×50 mL), dried over MgSO₄, filtered and roto-evaporated to yielda colorless oil (2.30 g, 90%). The crude product was purified by silicagel chromatography (50% EtOAc/Hexane, Rf=0.47) to yield a colorless oil(1.49 g, 58%).

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.88 (d, 1H, J=7.29Hz); 7.48 & 7.16 (m, 14H); 5.04 (m, 2H); 4.75 (m, 1H); 4.37 (d, 1H, 6.92Hz); 3.86 (s, 3H); 3.40 & 3.28 (m, 2H); 3.06 (m, 2H); 1.75, 1.61, & 1.32(m, 4H). ¹³C NMR (CDCl₃, 300 MHz): δ=171.61, 171.32, 171.23, 169.03,136.75, 135.89, 135.51, 133.89, 133.62, 131.14, 130.34, 129.77, 129.38,129.09, 129.00, 128.96, 128.88, 128.79, 127.48, 62.13, 60.90, 53.97,49.48, 38.10, 31.07, 24.48, 21.56. Mass Spectroscopy: (+FAB) 551 (M+H).

Example 54 Synthesis ofN-(3,5-Dichlorobenzenesulfonyl)-L-prolyl-L-phenylalanine

L-Proline methyl ester hydrochloride (2.68 g, 16.2 mmol) was dissolvedin pyridine (20 mL), 3,5-dichlorophenylsulfonyl chloride (3.57 g, 14.6mmol) was added and the mixture was stirred for 19 hr. Water (5 mL) wasadded and the mixture was stirred for 45 min before diluting with water(200 mL). The mixture was extracted with Et₂O (2×150 mL) and thecombined extracts were washed with water (3×100 mL), 1N HCl (150 mL) andsaturated aq NaHCO₃ (150 mL), then dried (MgSO₄), filtered andevaporated in vacuo to give 3,5-dichlorophenyl-sulfonyl-L-proline methylester as an oil (4.34 g, 88%).

The methyl ester was dissolved in MeOH (20 mL) and 1N NaOH (20 mL) wasadded. The mixture was gently warmed until homogeneous and then stirredfor 1.5 hr. The solvent was removed in vacuo and the residue taken up inwater (50 mL). The aqueous solution was washed with Et₂O (30 mL) andthen made acidic with 12 N HCl. The mixture was extracted with CHCl₃(2×40 mL) and the combined extracts were dried (MgSO₄), filtered andevaporated in vacuo to give N-(3,5-dichlorophenylsulfonyl)-L-proline asa solid (3.37 g, 83%).

3,5-Dichlorophenyl-sulfonyl-L-proline was coupled to L-phenylalanineethyl ester using the procedure described in Method 3 to give the ethylester of the title compound (348 mg, 45%).

The title compound was prepared via hydrolysis of the ethyl ester usingNaOH in MeOH. NMR analysis indicated that diastereomers were present.

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.11 (br s, 1H), 7.72 (d, 2H,J=1.9 Hz), 7.61 (q, 1H, J=2.0 Hz), 7.39-7.20 (6H), 4.92 (m, 1H), 4.15(m, 1H), 3.52-3.09 (4H), 2.11 (m, 1H), 1.95-1.49 (3H). ¹³C NMR (CDCl₃):δ=175.4, 175.2, 171.8, 171.7, 139.5, 139.3, 137.0, 136.6, 135.8, 134.0,134.0, 129.9, 129.5, 127.7, 126.7, 111.4, 63.1, 62.9, 53.8, 53.6, 50.5,50.3, 45.1, 37.9, 31.3, 30.8, 24.8, 24.6. Mass Spectroscopy: FAB m/e 471(M+H).

Example 55 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanineN-Hydroxysuccinimide Ester

Equimolar amounts of Cbz-L-phenylalanine, N-hydroxysuccinimide and DCCin CH₂Cl₂ were stirred for 3 hr at room temperature. Hexane was addedand the reaction mixture filtered through a bed of Celite. The filtratewas extracted with 10% citric acid, water and saturated NaCl, dried overMgSO₄, filtered and concentrated to yield the title compound as a solid,mp=186-188° C.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.66 (m, 3H); 7.28(m, 6H); 4.64 (m, 1H); 4.01 (m, 1H); 3.36 (m, 1H); 2.89 (m, 6H); 2.42(s, 3H); 1.90 & 1.68 (m, 3H); 1.33 & 1.15 (m, 4H). ¹³C NMR (CDCl₃, 300MHz): δ=173.07, 171.83, 166.89, 144.86, 137.90, 133.35, 130.51, 130.44,129.06, 128.33, 127.36, 63.21, 50.12, 47.74, 45.09, 38.30, 36.53, 34.30,30.67, 25.454, 24.35, 22.15. Mass Spectroscopy: (+FAB) 528 (M+H).

Example 56 Synthesis ofN-(Thiophene-2-sulfonyl)-L-prolyl-L-phenylalanine Methyl Ester

The title compound was prepared via Method 14 and isolated the as anoil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.63 (m, 1H);7.32-7.10 (br m, 7H); 4.85 (m, 1H); 4.09 (m, 1H); 3.77 (s, 3H); 3.40 (m,1H); 3.38 (m, 1H); 3.17 (m, 1H); 3.05 (m, 1H); 2.05 (m; 1H); 1.62-1.40(br m, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ=171.9, 170.9, 136.6, 136.2,133.9, 133.4, 129.8, 129.1, 128.3, 127.7, 63.2, 53.9, 53.1, 38.4, 30.1,24.8. Mass Spectroscopy: (PI-FAB) 423, (M+H)⁺.

Example 57 Synthesis ofN-(Thiophene-2-sulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared from Example 56 (285) via Method 6 andthe product was isolated as a solid.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.14 (d, 1H, J=8.0Hz); 8.02 (d, 1H, J=5.0 Hz); 7.70 (d, 1H, J=4.0 Hz); 7.25 (m, 6H); 4.51(m, 1H); 4.05 (m, 1H); 3.40 (m, 1H); 3.40 (m, 1H); 3.20-2.90 (br m, 3H);1.65-1.32 (br m, 4H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=172.9, 170.9, 137.8,136.4, 134.1, 133.4, 129.7, 128.6, 128.5, 126.8, 62.1, 53.5, 49.7, 36.8,30.7, 24.1. Mass Spectroscopy: (PI-FAB) 409, (M+H)⁺.

Example 58 Synthesis ofN-(1,3-Dimethyl-5-chloropyrazole-4-sulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared using Method 14 and Method 7, and theproduct was isolated as a white solid.

NMR data was as follows: ¹H NMR (CD₃OD, 300 MHz): δ=7.00-6.93 (br m,5H); 4.21 (m, 1H); 3.96 (m, 1H); 3.59 (s, 3H); 3.16-3.03 (br m, 3H);2.88 (m, 1H); 2.14 (s, 3H); 1.68 (m, 2H); 1.56-1.29 (br m, 2H). ¹³C NMR(CD₃OD, 75 MHz): δ=177.9, 173.6, 150.9, 139.7, 131.8, 131.4, 129.9,127.9, 115.3, 63.7, 57.6, 50.7, 39.4, 37.7, 32.6, 25.8, 14.9. MassSpectroscopy: (PI-FAB) 477, (M+H)⁺.

Example 59 Synthesis ofN-(1-Phenylethanesulfonyl)-L-prolyl-L-phenylalanine

PhCH₂CH₂SH was treated with Cl₂ in a rapidly stirred mixture of CHCl₃and H₂O, and then the CHCl₃ layer was washed with 5% NaHSO₃ and sat.NaCl, dried with MgSO₄, filtered, and concentrated to givePhCH₂CH₂SO₂Cl. This product was treated with Pro-OtBu and Et₃N in CH₂Cl₂to give, after aqueous workup, PhCH₂CH₂SO₂-Pro-OtBu. This product wastreated with HCO₂H, and the mixture was evaporated to givePhCH₂CH₂SO₂-Pro-OH. This product was treated with HCl.Phe-OtBu, EDAC,HOBT, and Et₃N in CH₂Cl₂ to give, after aqueous workup and flashchromatography, PhCH₂CH₂SO₂-Pro-Phe-OtBu. This product was treated withHCO₂H, and the mixture was evaporated to give the title compound as aclear oil.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=8.06 (d, J=8.1,1H), 7.32-7.14 (m, 10H), 4.52-4.24 (m, 1H), 4.26 (dd, J=7.0, J=2.6, 1H),3.40-3.31 (m, 3H), 3.29-3.23 (m, 2H), 3.08 (dd, J=13.8, J=4.8, 1H),2.97-2.90 (m, 3H), 2.05-1.99 (m, 1H), 1.77-1.65 (m, 3H). ¹³C NMR(DMSO-d₆, 75 MHz): δ=172.7, 171.4, 138.5, 137.4, 129.2, 128.6, 128.5,128.1, 126.5, 126.4, 60.8, 53.1, 50.0, 48.6, 36.6, 30.9, 28.6, 24.2.Mass Spectroscopy: (+FAB, 3-nitrobenzyl alcohol) 431 (MH+).

Example 60 Synthesis ofN-(1-Methylimidazole-4-sulfonyl)-L-prolyl-L-phenylalanine Methyl Ester

The title compound was prepared using Method 14 and was isolated as anoil.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=7.55-7.40 (br m,3H); 7.31-7.10 (br m, 5 H); 4.80 (m, 1H); 4.31 (m, 1H); 3.70 (s, 3H);3.49-3.18 (br m, 3H); 3.05 (m, 1 H); 2.05 (m, 1H); 1.74 (m, 2 H); 1.41(m, 1H). ¹³C NMR (CDCl₃, 75 MHz): δ=172.0, 171.6, 140.1, 137.9, 136.8,129.8, 129.0, 127.5, 126.1, 63.1, 53.9, 52.9, 50.3, 38.4, 34.6, 30.4,24.9. Mass Spectroscopy: (PI-FAB) 421, (M+H)⁺.

Example 61 Synthesis ofN-(1-Methylimidazole-4-sulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared from Example 60 (297) using Method 7 andwas isolated as a white solid.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=7.83 (d, 2H, J=9.0Hz), 7.65 (d, 1H, J=5.0 Hz), 7.20-7.05 (br m, 5H), 4.14 (m, 1H); 3.94(m, 1H); 3.67 (s, 3H); 3.04 (m, 4H), 1.81 (m, 1H), 1.50 (m, 2H); 1.29(m, 1H). ¹³C NMR (DMSO-d₆, 75 MHz): δ=173.1, 169.9, 140.7, 139.2, 136.0,130.0, 127.8, 126.7, 125.9, 63.0, 55.1, 49.7, 36.8, 33.9, 30.5, 23.9.Mass Spectroscopy: (PI-FAB) 429, (M+H)⁺.

Example 62 Synthesis ofN-(4-Amidinobenznesulfonyl)-L-prolyl-L-phenylalanine Methyl Ester

The title compound was prepared by refluxing the product from Example 64(313) (0.26 mmol) with ammonium acetate (0.32 mmol) in methanol (3 mL)for 3.5 hours and then concentrating under vacuum to give an oil (114mg, 96%). The crude product was purified on silica gel chromatography(100% EtOAc, Rf=0.01) to yield a colorless oil (40 mg, 34%).

NMR data was as follows: ¹H NMR (CD₃OD, 300 MHz): δ=7.77 (m, 4 H); 7.07(m, 5H); 4.48 (m, 1H); 4.01 (m , 1H); 3.51 (s, 3H); 3.25 (m, 1H); 3.04(m, 3H); 2.85 (m, 1H); 1.50 (m, 4H). ¹³C NMR (CD₃OD, 300 MHz): δ=174.45,173.58, 168.09; 143.77, 138.72, 134.54, 131.01, 130.84, 130.16, 130.11,128.57, 63.71, 55.72, 53.45, 51.09 38.72, 32.61, 25.97. MassSpectroscopy: (+FAB) 459 (M+H).

Example 63 Synthesis ofN-(4-Amidinobenzenesulfonyl)-L-prolyl-L-phenylalanine

The title compound was prepared from the product of Example 62 (303)using the procedure described in Method 7.

NMR data was as follows: ¹H NMR (CD₃OD, 300 MHz): δ=7.72 (m, 4H); 7.00(m, 5H); 4.51 (m, 0.5 H); 4.20 (t, 1H, J=5.92); 3.93 (m, 1H); 3.23 (m,0.5H); 3.14 (s, 1H); 3.04 (m, 1H); 2.85 (m, 1H); 1.48 (m, 4H). ¹³C NMR(CD₃OD, 300 MHz): δ=178.07, 173.31, 141.62, 141.36, 139.70, 138.57,131.36, 131.05, 131.00, 130.21, 130.17, 130.13, 130.02, 129.92, 129.78,129.71, 128.60, 128.03, 64.12, 57.64, 51.20, 39.52, 32.36, 25.88. MassSpectroscopy: (+FAB) 467 (M+H), 489 (M+Na).

Example 64 Synthesis ofN-(4-Thiomethoxyimidatylbenzenesulfonyl)-L-prolyl-L-phenylalanine MethylEster

The title compound was prepared by refluxing the product from Example 65(314) (0.20 mmoles) with acetone (3 mL) and methyl iodide (0.2 mL) for30 minutes, and concentrating the reaction under vacuum to yield ayellow oil (130 mg, <100%). The crude product was purified on silica gelchromatography (80% EtOAc/Hexane, Rf=0.49) to yield a yellow oil (110mg, 99%).

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=9.37 (bs, 2H); 8.11(d, 2H, J=8.43 Hz); 7.96 (d, 2H, J=8.49 Hz); 7.20 (m, 6H); 4.82 (m, 1H);4.11 (m, 1H); 3.74 (s, 3H); 3.41 (m, 1H); 3.23 (m, 1H); 3.09 (m, 2H);2.94 (s, 3H), 1.83 (m, 1H); 1.62 (m, 3H). ¹³C NMR (CDCl₃, 300 MHz):δ=186.47, 172.23, 171.65, 141.68, 136.53, 136.04, 130.32, 129.84,129.16, 129.12, 127.74, 62.98, 53.98, 53.33, 50.48, 38.21, 31.58, 31.15,24.83, 18.30. Mass Spectroscopy: (+FAB) 490 (M+H).

Example 65 Synthesis ofN-[4-(N-Methyl)thioamidobenzenesulfonyl]-L-prolyl-L-phenylalanine MethylEster

The title compound was prepared by bubbling hydrogen sulfide through asolution of the methyl ester of Example 29 (71) (0.3 mmoles) in pyridine(3 mL) and triethylamine (0.3 mL) at room temperature for 5 minutes. Theflask was sealed and the reaction stirred at room temperature for 18hours. The reaction mixture was concentrated under a stream of nitrogen.The residue was diluted with EtOAc (100 mL) and successively washed with2N KHSO₄ (2×50 mL) and saturated NaCl (50 mL), dried over MgSO₄,filtered and roto-evaporated to yield a yellow oil (146 mg, 100%).Silica gel TLC in 80% EtOAc/Hexane showed a single spot, Rf=0.06.

NMR data was as follows: ¹H NMR (CDCl₃, 300 MHz): δ=8.13 (d, 2H, J=11.54Hz); 7.94 (d, 2H, J=8.37 Hz); 7.69 (d, 2H, J=8.43 Hz); 7.20 (m, 6H);4.83 (m, 1H); 3.99 (d, 1H, J=6.17 Hz); 3.77 (s, 3H); 3.34 (t, 1H, J=6.59Hz); 3.25 (m, 1H); 3.03 (m, 1H); 1.87 (m, 1H); 1.45 (m, 5H). ¹³C NMR(CDCl₃, 300 MHz): δ=201.09, 172.29, 171.28, 144.50, 138.26, 136.37,129.76, 129.21, 128.71, 128.21, 127.82, 62.92, 53.74, 53.31, 50.20,38.37, 30.64, 30.54, 24.68, 21.68. Mass Spectroscopy: (+FAB) 476 (M+H).

Example 66

Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-D,L-β-(1,2,4-triazol-3-yl)alanine

An equimolar solution of β-(1,2,4-triazol-3-yl)-D,L-alanine methylester, N-(toluene-4-sulfonyl)]-L-proline hemibenzenate andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphatewith three equivalents of triethyl amine in acetonitrile (0.45 M) wasstirred at ambient temperature, under nitrogen, for 16 hr. The solventwas stripped off giving a dark reddish colored oil. The oil wasdissolved in ethyl acetate and washed with copious amounts of water,brine, and saturated sodium bicarbonate solution and once again withwater. The organic phase was dried (Na₂SO₄) and the solvent stripped offyielding a brown oil. The oil was hydrolyzed following the proceduredescribed in Method 7 yielding the title compound as a beige solid,mp=153-163° C.

NMR data was as follows: ¹H NMR (DMSO-d₆, 400 Mhz): δ=8.29 (s, 1H); 7.77(s, 1H); 7.57-7.52 (m, 2H); 7.37 (d, 1H, J=8.1 Hz); 7.32-7.29 (m, 2H);7.22-7.19 (m, 1H); 4.40 (dd, 1H, J=9.9 Hz, 4.2 Hz); 4.29-4.25 (m, 1H);3.95-3.87 (m, 1H); 3.82-3.77 (m, 1H); 3.15-3.06 (m, 1H); 2.96-2.85 (m,1H); 2.61-2.56 (m, 1H); 2.27 (s, 1H); 2.22 (s, 1H); 1.81 (t, 1H, J=7.7Hz); 1.75 (s, 3H); 1.75-1.50 (m, 1H); 1.17-1.15 (m, 1H); 1.05-0.94 (m,1H); 0.92-0.89 (m, 1H). IR (KBr, cm−1): 3400; 2960; 2850; 2500; 1775;1600; 1450; 1400; 1200; 1175; 1050; 1025; 875; 830; 775; 700; 600; 575;550. Mass Spectroscopy: (+FAB) 428.3 (M+Na); 406.3 (M−H); 319.4; 269.4;227.4; 205.4; 173.3; 113.12.

Example 67 Synthesis ofN-(Toluene-4-sulfonyl)-L-prolyl-D,L-β-(2-thiazolyl)alanine

The methyl ester of the title compound was prepared using the proceduredescribed in Method 3. The ester was hydrolyzed following the proceduredescribed in Method 6 to afford the title compound as a foam.

NMR data was as follows: ¹H NMR (DMSO-d₆, 400 MHz): δ=8.36 (d, 0.5H, J=8Hz); 8.24 (d, 0.5H, J=8 Hz); 7.7 (m, 3H); 7.58 (d, 1H, J=3 Hz); 7.4, (m,2H); 4.65 (m, 1H); 4.14 (m, 0.5H); 4.1 (m, 0.5H); 3.45 (m, 8H); 3.1 (m,1H); 2.39 (s, 3H); 1.75-1.4 (brd m, 4H). IR (KBr, cm⁻¹): 3425, 2900,1730, 1660, 1625, 1525, 1510, 1440, 1340, 1160, 1080, 660, 580, 550.Mass Spectroscopy: (+FAB) 424 ([M+H]⁺); 323; 279; 237; 215; 197; 181;149; 131; 109. HPLC (Primesphere C-18; 40/60 methanol/.01 M KH2PO4buffer pH=3.5; flow rate=1 ml/min): 16.6 min retention time diasteomer A(49%); 19.41 min retention time diasteomer B (51%).

Example 68 Synthesis ofN-[4-(3-Dimethylaminopropyloxy)-benzenesulfonyl]-L-prolyl-L-phenylalanine

4-(Methoxy)benzenesulfonyl chloride was dissolved in CH₂Cl₂ and chilledin an ice bath to 0° C. To this solution was added the hydrochloride ofPro-Phe-OMe (1 eq.) and TEA (2.2 eq). The reaction was allowed to warmto room temperature and stirred overnight under a stream of N₂. Thereaction mixture was then concentrated and the residue taken up in EtOAcand H₂O and the organic phase was washed with sat. NaHCO and brine,dried (MgSO₄), filtered, concentrated to a tacky solid and used withoutfurther purification. The solid product was treated with with BBr₃ (1 Min CH₂Cl₂, 3.0 eq., −78° C. for one hour then warmed to rt). Thereaction was then chilled in an ice bath and quenched with water.Following concentration, the residue was taken-up in EtOAc and water.The organic phase was washed with brine, dried (MgSO₄), filtered andconcentrated to a foam. The product was esterified with MeOH and HCl gas(0° C. to rt, 16 hours) and purified by column chromatography. Thisproduct was then added to a chilled (0° C.) THF solution oftriphenylphosphine (1.10 eq), 3-dimethylmino-1-propanol (1.0 eq) anddiethyl azodicarboxylate (1.10 eq). The reaction temperature was held at0° C. for 30 minutes and then allowed to warm to room tempetature andstirred overnight. The reaction mixture was concentrated and taken-up inEtOAc and washed with sat. NaHCO₃. The product was than extracted with0.2 N citric acid, and the aqueous phase washed with EtOAc. The aqueousphase was then made basic with solid NaHCO₃ and the product extractedwith EtOAc. The organic phase was washed with brine, dried (MgSO₄),filtered and concentrated. The product was purified by preparative TLC.The hydrolysis was performed using Method 7 to yield the title compoundas a mixture of diastereomers and was isolated as a white, hygroscopicsolid.

NMR data was as follows: ¹H NMR (DMSO-d₆, 300 MHz): δ=7.76 (d, 2H, J=9.0Hz); 7.74 (m, 1H); 7.15-7.10 (br m, 6 H); 4.07 (t, 2H, J=5.0 Hz); 3.90(m, 2H); 3.21-2.85 (m, 4H); 2.37 (m, 2H); 2.37 (t, 2H, J=7.0 Hz); 2.13(s. 6H); 1.85 (m, 2H); 1.69 (m, 1H); 1.50-1.25 (m, 3H). ¹³C NMR(DMSO-d₆, 75 MHz): δ=172.3, 169.7, 162.6, 139.3, 130.3, 130.1, 130.1,128.1, 127.9, 127.8, 125.9, 125.8, 115.3, 115.2, 66.7, 62.4, 55.8, 55.2,49.3, 45.5, 36.96, 30.6, 27.0, 23.9. Mass Spectroscopy: (PI−FAB) 548,(M−H+Na)⁺.

Example 69 Synthesis ofN-(4-Thiocarbamoylbenzenesulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanineBenzyl Ester

The title compound was prepared following the procedure of Example 65(314).

NMR data was as follows: ¹H NMR (CDCl₃): δ=8.48 (bs, 1H), 8.24 (bs, 1H),8.00-7.97 (d, 2H), 7.78-7.75 (d, 2H), 7.29-7.09 (m, 10H) 5.12 (m, 2H),4.48 (m, 1H), 4.49-4.46 (d, 1H), 4.334.30 (d, 1H), 3.09 (m, 2H),1.03-0.99 (d, 6H), 3.91 (s, 1H). ¹H NMR (CDCl₃): δ=200.5, 171.6, 169.2,144.3, 138.4, 136.2, 135.6, 130.0, 129.2, 128.8, 128.4, 127.8, 74.1,68.1, 55.1, 54.1, 51.1, 38.5, 29.8, 24.2, 21.7.

Example 70 Synthesis ofN-(4-Cyanobenzenesulfonyl)-L-(5,5-dimethyl)thiaprolyl-L-phenylalanineBenzyl Ester

The title compound was prepared following the procedure outlined for thepreparation of Example 29 (71).

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.95-7.92 (d, 2H), 7.77-7.74(d, 2H), 7.34-7.33 (m, 10H), 7.19-7.18 (m, 10H), 7.10-7.09 (m, 10H),6.97-6.95 (d, 1H), 5.15 (m, 2H), 4.91 (m, 1H), 4.47 (s, 2H), 3.10 (m,2H), 1.19-1.11 (2s, 6H). ¹C NMR (CDCl₃): δ=171.3, 168.4, 141.0, 136.3,135.6, 133.7, 130.0, 129.2, 127.7, 117.8, 74.1, 68.0, 55.3, 53.86, 51.1,38.7, 29.9, 24.4.

Example 71 Synthesis ofN-(Toluene-4-sulfonyl)-L-(thiamorpholyl-3-carbonyl)-L-phenylalanine

L-thiamorpholine-5-carboxylic acid was prepared by the method of Larssonand Carlson (Acta Chemica Scan. 48: 517-525 (1994)).N-(toluene-4-sulfonyl)-L-thiamorpholine-5-carboxylic acid was preparedusing the procedure described in Method 1. The title compound wasprepared according to the procedure described in Example 1 (9).

NMR data was as follows: ¹H NMR (CD₃OD): δ=7.49-7.04 (m, 9H), 4.62 (m,1H), 4.47 (m, 1H), 3.88 (m, 1H), 3.12-2.36 (m, 5H), 2.18 (s, 3H),2.18-1.92 (m, 5H). ¹³C NMR (CD₃OD): δ=175.1, 174.8, 170.8, 170.7, 146.2,146.1, 138.9, 138.5, 131.8, 131.1, 130.3, 129.1, 128.7, 57.0, 56.9,55.7, 45.5, 45.2, 38.9, 38.5, 28.6, 28.1, 26.8, 26.9, 22.3.

Example 72 Synthesis ofN-(Toluene-4-sulfonyl)-L-[(1,1-dioxo)thiamorpholyl-3-carbonyl]-L-phenylalanine

The title compound was prepared from the product of Example 71 (500)using the procedure described by Larsson and Carlson (Acta Chemica Scan.48, 522 (1994)).

NMR data was as follows: ¹H NMR (CD₃OD): δ=7.54 (m, 2H), 7.17-6.90 (m,10H), 5.07 (m, 1H), 4.45 (m, 1H), 4.08 (m, 1H), 3.8-3.3 (m, 2H),3.09-2.6 (m, 5H), 2.22 (s, 3H), 2.11 (s, 3H). ¹³C NMR (CD₃OD): δ=175.1,174.5, 168.5, 146.9, 139.5, 138.4, 137.8, 132.0, 131.1, 131.6, 130.2,129.2, 128.8, 126.9, 62.6, 57.8, 57.6, 55.7, 55.5, 51.6, 51.4, 43.8,43.6, 43.6, 38.7, 38.5, 22.2, 22.1.

Example 73 Synthesis ofN-(Toluene-4-sulfonyl)-L-[(1,1-dioxo)thiamorpholyl-3-carbonyl]-L-phenylalanineEthyl Ester

The title compound was prepared following the procedure outlined for thepreparation of Example 72 (501).

NMR data was as follows: ¹H NMR (CDCl₃): δ=7.72 (d, 1H), 7.63 (d, 1H),7.35 (m, 7H), 7.13 (m, 2H), 6.80 (d, 0.5H), 6.69 (d, 0.5H), 5.13 (m,0.5H), 5.00 (m, 0.5H), 4.89 (m, 0.5H), 4.77 (m, 0.5H), 4.21 (m, 2H),3.99 (m, 1H), 3.19 (m, 2H), 3.02 (m, 2H), 2.88 (m, 2H), 2.44 (s, 3H),1.25 (m, 3H). ¹³C NMR (CDCl₃): δ=171.5, 171.4, 165.4, 165.1, 146.1,136.1, 135.5, 131.2, 130.0; 130.0, 129.3, 129.2, 128.0, 127.8, 62.5,62.4, 56.8, 56.6, 54.2, 53.9, 50.2, 49.9, 49.4, 49.3, 42.6, 42.3, 38.1,37.8, 22.3, 14.7.

Example 74 Synthesis of N-(Toluene-4-sulfonyl)-L-prolyl-L-phenylalanine2-(1-Methyl-1,4-dihydropyridinyl-3-amido)ethyl Ester

A solution of(S,S)-1-methyl-3-[2-(3-phenyl-2-{[(1-toluene-4-sulfonyl)-pyrrolidine-2-carbonyl]-amino}-propionyloxy)-ethylcarbamoyl]-pyridiniumiodide (3.8 g) was prepared in nitrogen degassed water (1000 mL) andacetonitrile (55 mL). At ambient temperature, a mixture of Na₂S₂O₄ (2.82g) and NaHCO₃ (2.268 g) was added at once and the reaction mixturestirred another 3 hours at room temperature while being exposed to aslow nitrogen throughput. Thereafter, the aqueous phase was extractedwith chloroform (3×200 mL), and the organic extracts were combined,dried over MgSO₄, filtered and evaporated. The residue was flashchromatographed on 200 g silica gel. Elution with 2% methanol/chloroformgave 800 mg of the titled compound as, a solid, mp=68-72° C.

Other compounds prepared by the methods described above include thoseset forth in Table II below:

TABLE II

Q = —C(O)NR⁷— Example R¹ R² R³ R⁵ R^(6′) R⁷ No. n-butyl R²/R³ = cyclic—CH₂-φ —OH H 75 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —NH₂ H 76 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —O-ethyl H 77 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic 3 carbon atoms (L-pyrrolidinyl) —CH₂-φ

H 78 p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₃ H 79 —CH₂S—C(CH₃)₂-(L-5,5-dimethylthiazolidin- 4-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ OH H 80—CH₂CH(OCH₃)CH₂- (3-methoxy-L-pyrrolidin-2- yl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ 2-β-isopropyl-4-α- H 81 3 carbon atoms methyl- (L-pyrrolidinyl)cyclohex-1-oxy p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₂CH₂— H 82 3 carbonatoms NHC(O)-pyrid-3-yl (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ—OCH₂CH₂— H 83 3 carbon atoms NHC(O)— (L-pyrrolidinyl)N-methylpyrid-3-yl p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —O-cholest-5-en-3- H84 3 carbon atoms β-yl (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ—OCH₂CH₂— H 85 3 carbon atoms NHC(O)—N-methyl- (L-pyrrolidinyl)1,4-dihydro-pyrid- 3-yl p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH Q = —C(S)NH—86 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H87 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OH H88 —CH₂—CH₂—C(CH₃)₂— (L-4,4-dimethylpyrrolidinyl) p-CH₃-φ- R²/R³ =cyclic —CH₂-φ —OH H 89 —CH₂—SO₂—C(CH₃)₂— (L-1,1-dioxo-5,5-dimethylthiazolidin-4-yl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OC(CH₃)₃ H 903 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic —CH₂-φ —OCH₃ Q =—C(S)NH— 91 3 carbon atoms (L-pyrrolidinyl) φ- R²/R³ = cyclic —CH₂-φ—OCH₃ H 92 3 carbon atoms (L-pyrrolidinyl) p-CH₃-φ- R²/R³ = cyclic—CH₂-φ —OCH₂CH₃ H 93 —C(O)—CH₂—CH₂— (L-5-oxopyrrolidinyl) p-CH₃-φ- R²/R³= cyclic —CH₂-φ —OH H 94 —C(O)—CH₂—CH₂— (L-5-oxopyrrolidinyl)

Example 95 In Vitro Assay for Determining Binding of Candidate Compoundsto VLA-4

An in vitro assay was used to assess binding of candidate compounds toα₄β₁ integrin. Compounds which bind in this assay can be used to assessVCAM-1 levels in biological samples by conventional assays (e.g.,competitive assays). This assay is sensitive to IC₅₀ values as low asabout 1 nM.

The activity of α₄β₁ integrin was measured by the interaction of solubleVCAM-1 with Jurkat cells (e.g., American Type Culture Collection Nos.TIB 152, TIB 153, and CRL 8163), a human T-cell line which expresseshigh levels of α₄β₁ integrin. VCAM-1 interacts with the cell surface inan α₄β₁ integrin-dependent fashion (Yednock, et al. J. Biol. Chem. 270:28740 (1995)).

Recombinant soluble VCAM-1 was expressed as a chimeric fusion proteincontaining the seven extracellular domains of VCAM-1 on the N-terminusand the human IgG₁ heavy chain constant region on the C-terminus. TheVCAM-1 fusion protein was made and purified by the manner described byYednock, supra.

Jurkat cells were grown in RPMI 1640 supplemented with 10% fetal bovineserum, penicillin, streptomycin and glutamine as described by Yednock,supra.

Jurkat cells were incubated with 1.5 mM MnCl₂ and 5 μg/mL 15/7 antibodyfor 30 minutes on ice. Mn⁺² activates the receptor to enhance ligandbinding, and 15/7 is a monoclonal antibody that recognizes anactivated/ligand occupied conformation of α₄β₁ integrin and locks themolecule into this conformation, thereby stabilizing the VCAM-1/α₄β₁integrin interaction. Yednock, et al., supra. Antibodies similar to the15/7 antibody have been prepared by other investigators (Luque, et al.,J. Biol. Chem. 271:11067 (1996)) and may be used in this assay.

Cells were then incubated for 30 minutes at room temperature withcandidate compounds, in various concentrations ranging from 66 μM to0.01 μM using a standard. 5-point serial dilution. 15 μL solublerecombinant VCAM-1 fusion protein was then added to Jurkat cells andincubated for 30 minutes on ice. (Yednock et al., supra.).

Cells were then washed two times and resuspended in PE-conjugated goatF(ab′)₂ anti-mouse IgG Fc (Immunotech, Westbrook, Me.) at 1:200 andincubated on ice, in the dark, for 30 minutes. Cells were washed twiceand analyzed with a standard fluorescence activated cell sorter (“FACS”)analysis as described in Yednock, et al., supra.

Compounds having an IC₅₀ of less than about 15 μM possess bindingaffinity to α₄β₁.

When tested in this assay, each of the compounds in Examples 1-91 has anIC₅₀ of 15 μM or less.

Example 96 In Vitro Saturation Assay for Determining Binding ofCandidate Compounds to α₄β₁

The following describes an in vitro assay to determine the plasma levelsneeded for a compound to be active in the Experimental AutoimmuneEncephalomyelitis (“EAE”) model, described in the next example, or inother in vivo models.

Log-growth Jurkat cells are washed and resuspended in normal animalplasma containing 20 μg/ml of the 15/7 antibody (described in the aboveexample).

The Jurkat cells are diluted two-fold into either normal plasma samplescontaining known candidate compound amounts in various concentrationsranging from 66 μM to 0.01 μM, using a standard 12 point serial dilutionfor a standard curve, or into plasma samples obtained from theperipheral blood of candidate compound-treated animals.

Cells are then incubated for 30 minutes at room temperature and washedtwice with phosphate-buffered saline (“PBS”) containing 2% fetal bovineserum and 1 mM each of calcium chloride and magnesium chloride (assaymedium) to remove unbound 15/7 antibody.

The cells are then exposed to phycoerythrin-conjugated goat F(ab′)anti-mouse IgG Fc (Immunotech, Westbrook, Me.), which has been adsorbedfor any non-specific cross-reactivity by co-incubation with 5% serumfrom the animal species being studied, at 1:200 and incubated in thedark at 4° C. for 30 minutes.

Cells are washed twice with assay medium and resuspended in the same.They are then analyzed with a standard fluorescence activated cellsorter (“FACS”) analysis as described in Yednock et al., J. Biol. Chem.270: 28740 (1995).

The data is then graphed as fluorescence versus dose, e.g., in a normaldose-response fashion. The dose levels that result in the upper plateauof the curve represent the levels needed to obtain efficacy in an invivo model.

This assay may also be used to determine the plasma levels needed tosaturate the binding sites of other integrins, such as the α₉β₁integrin, which is the integrin most closely related to a α₄β₁ (Palmeret al., J. Cell Bio. 123: 1289 (1993)). Such binding is predictive of invivo utility for inflammatory conditions mediated by α₉β₁ integrin,including, by way of example, airway hyper-responsiveness and occlusionthat occurs with chronic asthma, smooth muscle cell proliferation inatherosclerosis, vascular occlusion following angioplasty, fibrosis andglomerular scarring as a result of renal disease, aortic stenosis,hypertrophy of synovial membranes in rheumatoid arthritis, andinflammation and scarring that occur with the progression of ulcerativecolitis and Crohn's disease.

Accordingly, the above-described assay may be performed with a humancolon carcinoma cell line, SW 480 (ATTC #CCL228), transfected with cDNAencoding α₉ integrin (Yokosaki et al., J. Biol. Chem. 2: 26691 (1994)),in place of the Jurkat cells, to measure the binding of the α₉β₁integrin. As a control, SW 480 cells which express other α and β₁subunits may be used.

Accordingly, another aspect of this invention is directed to a methodfor treating a disease in a mammalian patient, which disease is mediatedby α₉β₁, and which method comprises administering to said patient atherapeutically effective amount of a compound of this invention. Suchcompounds are preferably administered in a pharmaceutical compositiondescribed herein above. Effective daily dosing will depend upon the age,weight and condition of the patient, which factors can be readilyascertained by the attending clinician. However, in a preferredembodiment, the compounds are administered from about 20 to 500 μg/kgper day.

Example 97 In Vivo Evaluation—Experimental Autoimmune Encephalomyelitis

The standard multiple sclerosis model, Experimental Autoimmune (orAllergic) Encephalomyelitis (“EAE”), was used to determine the effect ofcandidate compounds to reduce motor impairment in rats or guinea pigs.Reduction in motor impairment is based on blocking adhesion betweenleukocytes and the endothelium and correlates with anti-inflammatoryactivity in the candidate compound. This model has been previouslydescribed by Keszthelyi et al., Neurology 47:1053-1059 (1996), andmeasures the delay of onset of disease.

Brains and spinal cords of adult Hartley guinea pigs were homogenized inan equal volume of phosphate-buffered saline. An equal volume ofFreund's complete adjuvant (100 mg mycobacterium tuberculosis plus 10 mlFreund's incomplete adjuvant) was added to the homogenate. The mixturewas emulsified by circulating it repeatedly through a 20 ml syringe witha peristaltic pump for about 20 minutes.

Female Lewis rats (2-3 months old, 170-220 g) or Hartley guinea pigs (20day old, 180-200 g) were anesthetized with isoflurane and threeinjections of the emulsion, 0.1 ml each, were made in each flank. Motorimpairment onset is seen in approximately 9 days.

Candidate compound treatment began on Day 8, just before onset ofsymptoms. Compounds were administered subcutaneously (“SC”), orally(“PO”) or intraperitoneally (“IP”). Doses were given in a range of 10mg/kg to 200 mg/kg, bid, for five days, with typical dosing of 10 to 100mg/kg SC, 10 to 50 mg/kg PO, and 10 to 100 mg/kg IP.

Antibody GG5/3 against α₄β₁ integrin (Keszthelyi et al., Neurology47:1053-1059 (1996)), which delays the onset of symptoms, was used as apositive control and was injected subcutaneously at 3 mg/kg on Day 8 and11.

Body weight and motor impairment were measured daily. Motor impairmentwas rated with the following clinical score:

0 no change 1 tail weakness or paralysis 2 hindlimb weakness 3 hindlimbparalysis 4 moribund or dead

A candidate compound was considered active if it delayed the onset ofsymptoms, e.g., produced clinical scores no greater than 2 or slowedbody weight loss as compared to the control.

When tested in this in vivo assay, at least the compound of Example 3was active.

Example 98 In Vivo Evaluation—Asthma

Inflammatory conditions mediated by α₄β₁ integrin include, for example,airway-hyper-responsiveness and occlusion that occurs with chronicasthma. The following describes an asthma model which can be used tostudy the in vivo effects of the compounds of this invention for use intreating asthma.

Following the procedures described by Abraham et al, J. Clin. Invest.93:776-787 (1994) and Abraham et al, Am J. Respir Crit Care Med.156:696-703 (1997), both of which are incorporated by reference in theirentirety, compounds of this invention are formulated into an aerosol andadministered to sheep which are hypersensitive to Ascaris suum antigen.Compounds which decrease the early antigen-induced bronchial responseand/or block the late-phase airway response, e.g., have a protectiveeffect against antigen-induced late responses and airwayhyper-responsiveness (“AHR”), are considered to be active in this model.

Allergic sheep which are shown to develop both early and late bronchialresponses to inhaled Ascaris suum antigen are used to study the airwayeffects of the candidate compounds. Following topical anesthesia of thenasal passages with 2% lidocaine, a balloon catheter is advanced throughone nostril into the lower esophagus. The animals are then intubatedwith a cuffed endotracheal tube through the other nostril with aflexible fiberoptic bronchoscope as a guide.

Pleural pressure is estimated according to Abraham (1994). Aerosols (seeformulation below) are generated using a disposable medical nebulizerthat provides an aerosol with a mass median aerodynamic diameter of 3.2μm as determined with an Andersen cascade impactor. The nebulizer isconnected to a dosimeter system consisting of a solenoid valve and asource of compressed air (20 psi). The output of the nebulizer isdirected into a plastic T-piece, one end of which is connected to theinspiratory port of a piston respirator. The solenoid valve is activatedfor 1 second at the beginning of the inspiratory cycle of therespirator. Aerosols are delivered at V_(T) of 500 ml and a rate of 20breaths/minute. A 0.5% sodium bicarbonate solution only is used as acontrol.

To assess bronchial responsiveness, cumulative concentration-responsecurves to carbachol can be generated according to Abraham (1994).Bronchial biopsies can be taken prior to and following the initiation oftreatment and 24 hours after antigen challenge. Bronchial biopsies canbe preformed according to Abraham (1994).

An in vitro adhesion study of alveolar macrophages can also be performedaccording to Abraham (1994), and a percentage of adherent cells iscalculated.

Aerosol Formulation

A solution of the candidate compound in 0.5% sodium bicarbonate/saline(w/v) at a concentration of 30.0 mg/mL is prepared using the followingprocedure:

A. Preparation of 100.0 mL of 0.5% Sodium Bicarbonate/Saline StockSolution

Ingredient Gram/100.0 μmL Final Concentration Sodium Bicarbonate 0.5 g     0.5% Saline q.s. ad 100.0 mL q.s. ad 100%

Procedure:

1. Add 0.5 g sodium bicarbonate into a 100 mL volumetric flask.

2. Add approximately 90.0 mL saline and sonicate until dissolved.

3. Q.S. to 100.0 mL with saline and mix thoroughly.

B. Preparation of 30.0 mg/mL Candidate Compound: 10.0 mL

Ingredient Gram/10.0 mL Final Concentration Candidate Compound 0.300 g30.0 mg/mL 0.5% Sodium Bicarbonate/ q.s. ad 10.0 mL q.s ad 100% SalineStock Solution

Procedure:

1. Add 0.300 g of the candidate compound into a 10.0 mL volumetricflask.

2. Add approximately 9.7 mL of 0.5% sodium bicarbonate/saline stocksolution.

3. Sonicate until the candidate compound is completely dissolved.

4. Q.S. to 10.0 mL with 0.5% sodium bicarbonate/saline stock solutionand mix thoroughly.

Using a conventional oral formulation, compounds of this invention wouldbe active in this model.

What is claimed is:
 1. A compound of formula I:

where R¹ is selected from the group consisting of alkyl, substitutedalkyl, phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl,heterocyclic, substituted heterocyclic, heteroaryl and substitutedheteroaryl; R² and R³, together with the nitrogen atom bound to R² andthe carbon atom bound to R³, form a saturated heterocyclic group or asaturated substituted heterocyclic group with the proviso that whenmonosubstituted, the substituent on said saturated heterocyclic group isnot carboxyl; R⁵ is selected from the group consisting of—(CH₂)_(n)-aryl and —(CH₂)_(n)-heteroaryl, where n is an integer equalto 1 to 4; Q is —C(X)NR⁷— wherein R⁷ is selected from the groupconsisting of hydrogen and alkyl, and X is selected from the groupconsisting of oxygen and sulfur; and pharmaceutically acceptable saltsthereof with the proviso that when R¹ is 2,4,6-trimethylphenyl, R² andR³ together with the pendent nitrogen and carbon atoms form apyrrolidinyl ring and Q is —C(O)NH—, then R⁵ is not benzyl.
 2. Acompound of formula II:

where R¹ is selected from the group consisting of alkyl, substitutedalkyl, phenyl, substituted phenyl, cycloalkyl, heterocyclic, substitutedheterocyclic, and heteroaryl; R² and R³, together with the nitrogen atombound to R² and the carbon atom bound to R³, form a saturatedheterocyclic group or a saturated substituted heterocyclic group withthe proviso that when monosubstituted, the substituent on said saturatedheterocyclic group is not carboxyl; R⁵ is selected from the groupconsisting of —(CH₂)_(n)-aryl and —(CH₂)_(n)-heteroaryl, where n is aninteger equal to 1 to 4; R⁶ is selected from the group consisting ofamino, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy,—O-(N-succinimidyl), —NH-adamantyl, —O-cholest-5-en-3-β-yl, —NHOY whereY is hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl,—NH(CH₂)_(p)COOY where p is an integer of from 1 to 8 and Y is asdefined above, —OCH₂NR⁹R¹⁰ where R⁹ is selected from the groupconsisting of —C(O)-aryl and —C(O)-substituted aryl and R¹⁰ is selectedfrom the group consisting of hydrogen and —CH₂COOR¹¹ where R¹¹ is alkyl,and —NHSO₂Z where Z is alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic and substituted heterocyclic; Q is —C(X)NR⁷— wherein R⁷ isselected from the group consisting of hydrogen and alkyl, and X isselected from the group consisting of oxygen and sulfur; andpharmaceutically acceptable salts thereof, with the following provisos:A. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a pyrrolidin-2-yl ring, R⁵ is benzyl andQ is —C(O)NH—, then R⁶ is not —NH(CH₂)₂CO₂Et or -(1R,2S,5R)-(−)-menthylester; B. when R¹ is 4-methylphenyl, R² and R³ together with the pendentnitrogen and carbon atoms form a 3-β-phenyl-D-pyrrolidin-2-yl ring, R⁵is benzyl and Q is —C(O)NH—, then R⁶ is not —OCH₂CH₃; C. when R¹ is4-methylphenyl, R² and R³ together with the pendent nitrogen and carbonatoms form a pyrrolidin-2-yl ring, R⁵ is D-benzyl and Q is —C(O)NH—,then R⁶ is not —OCH₂CH₃; and D. when R¹ is 4-methylphenyl, R² and R³together with the pendent nitrogen and carbon atoms form a5,5-dimethyl-1,1-dioxo-thiaprolyl ring, R⁵ is benzyl and Q is —C(O)NH—,then R⁶ is not —OC(CH₃)₃.
 3. The compound according to claims 1 or 2,wherein R¹ is selected from the group consisting of phenyl andsubstituted phenyl.
 4. The compound according to claims 1 or 2, whereinR¹ is selected from the group consisting of 4-methylphenyl, methyl,benzyl, n-butyl, 4-chlorophenyl, 4-methoxyphenyl, phenyl,2,4,6-trimethylphenyl, 2-(methoxycarbonyl)phenyl, 2-carboxyphenyl,3,5-dichlorophenyl, 4-trifluoromethylphenyl, 3,4-dichlorophenyl,3,4-dimethoxyphenyl, 4-(CH₃C(O)NH—)phenyl, 4-trifluoromethoxyphenyl,4-cyanophenyl, isopropyl, 3,5-di-(trifluoromethyl)phenyl,4-t-butylphenyl, 4-t-butoxyphenyl, 4-nitrophenyl, 2-thienyl, phenethyl,4-bromophenyl,4-amidinophenyl, 4-methylamidinophenyl,4-[CH₃SC(═NH)]phenyl, 2-thiazolyl, 4-[H₂NC(S)]phenyl, 4-aminophenyl,4-fluorophenyl, 2-fluorophenyl, 3-fluorophenyl, 3,5-difluorophenyl,pyridin-3-yl, pyrimidin-2-yl, and 4-(3′-dimethylamino-n-propoxy)-phenyl.5. The compound according to claims 1 or 2, wherein R² and R³ togetherwith the nitrogen atom bound to the R² substituent and the carbon boundto the R³ substituent form a heterocyclic or substituted heterocyclicgroup of 4 to 6 ring atoms having 1 to 2 heteroatoms in the ringselected from the group consisting of nitrogen, oxygen and sulfur, whichring is optionally substituted with 1 to 2 substituents selected fromthe group consisting of fluoro, methyl, hydroxy, amino, phenyl,thiophenyl and thiobenzyl, or can be fused to another saturatedheterocyclic or cycloalkyl ring such as a cyclohexyl ring to provide fora fused ring heterocycle of from 10 to 14 ring atoms having 1 to 2heteroatoms in the ring selected from the group consisting of nitrogen,oxygen and sulfur.
 6. A compound according to claims 1 or 2, wherein theR² and R³ heterocyclic ring is selected from the group consisting ofazetidin-2-yl, thiazolidin-4-yl, piperidin-2-yl, piperizin-2-yl,thiomorpholin-3-yl and pyrrolidin-2-yl.
 7. The compound according toclaims 1 or 2, wherein the R² and R³ substituted heterocyclic ring isselected from the group consisting of 4-hydroxypyrrolidin-2-yl,4-fluoropyrrolidin-2-yl, 3-phenylpyrrolidin-2-yl,3-thiophenylpyrrolidin-2-yl, 4-aminopyrrolidin-2-yl,3-methoxypyrrolidin-2-yl, 4,4-dimethylpyrrolidin-2-yl,4-N-Cbz-piperizin-2-yl, 5,5-dimethyl-thiazolidin-4-yl,1,1-dioxo-thiazolidin-4-yl, L-1,1-dioxo-5,5-dimethylthiazolidin-4-yl and1,1-dioxothiomorpholinyl.
 8. The compound according to claims 1 or 2,wherein R⁵ is selected from the group consisting of benzyl, phenethyl,—CH₂-(3-indolyl), —CH₂-(1-naphthyl), —CH₂-(2-naphthyl),—CH₂-(2-thienyl), —CH₂-(3-pyridyl), —CH₂-(5-imidazolyl),—CH₂-3-(1,2,4-triazolyl) and —CH₂-(2-thiazolyl).
 9. The compoundaccording to claim 2, wherein R⁶ is selected from the group consistingof methoxy, ethoxy, iso-propoxy, n-butoxy, t-butoxy, cyclopentoxy,neo-pentoxy, 2-α-iso-propyl-4-β-methylcyclohexoxy,2-β-isopropyl-4-β-methylcyclohexoxy, —NH₂, benzyloxy, —NHCH₂COOH,—NHCH₂CH₂COOH, —NH-adamantyl, —NHCH₂CH₂COOCH₂CH₃, —NHSO₂-p-CH₃-φ, —NHOR⁸where R⁸ is hydrogen, methyl, iso-propyl or benzyl, O-(N-succinimidyl),—O-cholest-5-en-3-β-yl, —OCH₂—OC(O)C(CH₃)₃, —O(CH₂)_(z)NHC(O)W where zis 1 or 2 and W is selected from the group consisting of pyrid-3-yl,N-methylpyridyl, and N-methyl-1,4-dihydro-pyrid-3-yl and —NR″C(O)-R′where R′ is aryl, heteroaryl or heterocyclic and R″ is hydrogen or—CH₂C(O)OCH₂CH₃.
 10. A method for binding VLA-4 in a biological samplewhich method comprises contacting the biological sample with a compoundaccording to claim 1 or 2 under conditions wherein said compound bindsto VLA-4.
 11. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a therapeutically effective amount of one or moreof the compounds of claims 1 or
 2. 12. A method for the treatment of aninflammatory disease in a patient mediated by VLA-4 which methodcomprises administering to the patient the pharmaceutical composition ofclaim
 11. 13. The method according to claim 12 wherein said inflammatorydisease is selected from the group consisting of asthma, Alzheimer'sdisease, atherosclerosis, AIDS dementia, diabetes (including acutejuvenile onset diabetes), inflammatory bowel disease (includingulcerative colitis and Crohn's disease), multiple sclerosis, rheumatoidarthritis, tissue transplantation, tumor metastasis, meningitis,encephalitis, stroke and other cerebral traumas, nephritis, retinitis,atopic dermatitis, psoriasis, myocardial ischemia and acuteleukocyte-mediated lung injury such as that which occurs in adultrespiratory distress syndrome.